Ion triggered alkylation of biological targets by silyloxy aromatic agents

ABSTRACT

A silyloxy aromatic derivative capable of alkylating a target biological molecule when activated by ionic strength. A sequence directed reagent may be constructed by conjugating a methyl silyloxy aromatic derivative to a hexamethyiamino linker attached to either the 5&#39; or 3&#39; terminus of an oligonucleotide. Annealing this modified fragment of DNA to its complementary sequence allows for target modification subsequent to ionic activation. The product of this reaction is a covalent crosslink between the reagent and target strands resulting from an alkylation of DNA by the activated silyloxy aromatic derivative. In a preferred embodiment, a nitrophenyl or bromo group is attached to a methyl group of the silyloxy aromatic derivative. This reagent may be similarly linked to an oligonucleotide probe. Activation of the alkylating agent by an ionic signal (X) which may naturally occur, or may be introduced into the media containing the target molecule, such as by the introduction of a salt (MX).

This application is a continuation-in-part of application Ser. No.07/606,463, filed Oct. 31, 1990, now U.S. Pat. No. 5,296,350.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to silyloxy aromatic alkylating agents thatoptionally include a probe capable of associating with biologicaltargets. The alkylating agents are activated for reaction by ionicstrength.

2. Background of the Related Art

Currently prescribed chemotherapeutic agents acting at the level of DNAare often effective, but their therapeutic index is quite poor, limitedby the lack of target specificity. An international research effort hasbeen underway using a wide range of techniques to develop a genespecific drug--a "magic bullet" that is aimed at a single DNA sequencewithin a cell.

The technological advances allowing for facile DNA synthesis haveproduced innumerable protocols which rely on custom oligonucleotides,used as probes to screen for complementary sequences within plasmids,chromosomes and DNA libraries. See, for example, Landegren et al., "DNADiagnostics-Molecular Techniques and Automation", Science, 242, 229(1988). The specificity of oligonucleotide hybridization has beenutilized for "antisense" methods controlling selective expression ofgenes both in vivo and in vitro. For example, see Miller et al.,"Oligonucleotide Inhibitors of Gene Expression in Living Cells: NewOpportunities in Drug Design", Ann. Reports in Med. Chem., 23, 295(1988). Sequence recognition by the binding of probes most often dependson only the non-covalent forces of hydrogen bonding formed betweencomplementary base pairs. Complexation of this type is quite sufficientfor many applications, but covalent cross-linking of duplex structurescould simplify many of the current protocols and provide newopportunities for processing DNA in a sequence specific manner.Messenger RNA has become a viable target for inhibiting the expressionof a desired gene in vivo. See, for example, Toulme et al.,"Antimessenger Oligodeoxyribo-Nucleotides: An Alternative to AntisenseRNA for Artificial Regulation of Gene Expression--A Review", Gene, 72,51-58 (1988); and, Stein et al., "Oligodeoxyribo-Nucleotides asInhibitors of Gene Expression: A Review", Cancer Research, 48, 2659-2668(1988). Compounds created for this selective reaction have drawn fromthe advances in site specific modification of DNA. For example, seeBarton, "Metals and DNA: Molecular Left-Handed Complements", Science,233, 727-734 (1986), and Dervan, "Design of Sequence-SpecificDNA-Binding Molecules", Science, 232, 464-471 (1986).

Use of such compounds also depends on the synthesis of metabolicallystable oligonucleotides that can traverse cell membranes. For example,see Blake et al., "Hybridization Arrest of Globin Synthesis in RabbitReticulocyte Lysates and Cells by OligodeoxyribonucleosideMethylphosphonates", Biochemistry, 24, 6139-6145 (1985). Also, seeAgrawal et al., "Oligodeoxynucleoside phosphoramidates andphosphorothioates as inhibitors of human immunodeficiency virus", Proc.Natl. Acad. Sci. U.S.A., 85, 7079-7083 (1988).

Only recently introduced, the technique of oligonucleotide-directedirreversible DNA modification holds great potential as an in vitro toolfor molecular biologists. See, for example, Dervan, "Design ofSequence-Specific DNA-Binding Molecules", Science, 232, 464-471 (1986);and Iverson et al., in "Non-enzymatic Sequence-Specific Cleavage ofSingle-Stranded DNA to Nucleotide Resolution. DNA Methyl ThioletherProbes", J. Am. Chem. Soc., 109, 1241-1243 (1987). Site specificity isenforced by the hybridization of the oligomer-reactant to its complementsequence prior to reagent action. Target selectivity can then beconferred, in theory, to most reactive compounds by attaching them tooligonucleotides. The required prehybridization step, however, generallylimits this technique's applicability to accessible single strandpolynucleotide targets or duplex probes when triple helical formation ispossible, see Maher III et al., "Inhibition of DNA Binding Proteins byOligonucleotide-Directed Triple Helix Formation", Science, 245, 725-730(1989); Science 245, 967-971 (1989); and Science, 249, 73-75 (1990).

Site-directed covalent modification is also constrained by the nature ofthe reactive group incorporated into the oligomer. Although a largenumber of reactive appendages are available for related use in vitro, asreported by Iverson et al., J. Am. Chem. Soc., 109 (1987) supra; and byDervan, Science, 232 (1986) supra, only a limited set of these may applyin a controlled activated manner, either in vitro or for in vivo use.

Sequence recognition between synthetic oligonucleotides andmacromolecular DNA represent the keystone of numerous techniquesrequired in molecular biology. For example, see Symons, Nucleic AcidProbes, CRC Press, Inc., Boca Raton, Fla. (1989). The fidelity of thisprocess is typically determined only by the hydrogen bonds formedbetween complementary bases of double and triple helical DNA. Suchassociations are sufficient for most applications, but covalentstabilization of a target-probe complex could simplify a variety ofprotocols including those used to diagnose genetic, malignant andinfectious diseases; e.g., see discussions by Landegren et al.,"DNA-Diagnostic Molecular Techniques and Automation", Science 242, 229(1988); and Gamper et al., "Reverse Southern Hybridization" NucleicAcids Research, 14, 9943 (1986).

A general method for this cross-linking has been demonstrated with theconstruction of oligonucleotide-directed alkylating agent, reported byKnorre et al., "Complementary-Addressed (Sequence-Specific) Modificationof Nucleic Acids", Prog. Nucleic Acids Res. Mol. Biol., 32, 291 (1985);Webb and Matteucci, "Sequence-Specific Crosslinking ofDeoxyoligonucleotides via Hybridization-Triggered Alkylation", J. Am.Chem. Soc., 108, 2764 (1986); Dervan, "Design of Sequence-SpecificDNA-binding Molecules", Science 232, 464 (1986); and Meyer et al.,"Efficient, Specific Crosslinking and Cleavage of DNA by Stable,Synthetic Complementary Oligonucleotides", J. Am. Chem. Soc., 111, 8517(1989). However, limitations are placed on these reagents because oftheir inherent reactivity. Only mildly reactive species would allow fortarget recognition to precede covalent modification. An alternativeapproach has relied on moieties that remain inert until triggered by achemical or photochemical signal. For example, see Van Houten et al.,"Action Mechanism of ABC Excision Nuclease on a DNA Substrate Containinga Psoralen Crosslink at a Defined Position", Proc. Natl. Acad. Sci.U.S.A., 83, 8077 (1986); Lee et al., "Interaction ofPsoralen-Derivatized Oligodeoxyribonucleoside Methylphosphonates withSingle-Stranded DNA", Biochemistry, 27, 3197 (1988); Iverson et al.,"Nonenzymatic Sequence-Specific Cleavage of Single-Stranded DNA toNucleotide Resolution. DNA Methyl Thiolether Probes", J. Am. Chem. Soc.,109, 1241 (1987); Chatterjee and Rokita, "Inducible Alkylation of DNAUsing an Oligonucleotide-Quinone Conjugate", J. Am. Chem. Soc. 112, 6397(1990); and also see co-pending patent application U.S. Ser. No.07/442,947, filed on Nov. 29, 1989 the disclosure of which isincorporated by reference herein.

Organosilane compounds have been used as intermediates in the formationof quinone methides in aprotic solvents. For example, see Ramage et al.,"Solid Phase Peptide Synthesis: Fluoride Ion Release of Peptide from theResin", Tet. Lett., 28, 4105 (1987); Mullen and Barany, "A NewFluoridolyzable Anchoring Linkage for Orthogonal Solid Phase PeptideSynthesis", J. Org. Chem. 53, 5240 (1988); Trahanovsky et al.,"Observation of Reactive o-Quinodimethanes by Flow NMR", J. Am. Chem.Soc., 110, 6579 (1988); and Angle and Turnbull, "p-Quinone MethideInitiated Cyclization Reactions", J. Am. Chem. Soc., 111, 1136 (1989).

Yabusaki et al., in PCT Published Application No. WO 85/02628, describecross-linking agents for binding an oligonucleotide probe to a targetDNA or RNA molecule. Three types of cross-linking agents are described,including "bi-functional photoreagents", "mixed chemical and biochemicalbifunctional reagents" and "bifunctional chemical cross-linkingmolecules". The bifunctional photoreagents contain two photochemicallyreactive sites that bind covalently to the probe and to the targetmolecules. The mixed chemical and photochemical bifunctional reagent isbound non-photochemically to the probe molecule, followed byphotochemical binding to the target molecule. Non-photochemical bindingis described as a chemical reaction such as alkylation, condensation oradditional. Bi-functional chemical cross-linking molecules are said tobe activated either catalytically or by high temperature followinghybridization.

Although Yabusaki et al. generally hypothesize the concept of abifunctional photochemical reagent and a mixed chemical andphotochemical reagent, there is no specific description of thesemolecules. All of the reagents they describe are well knownphotochemical reagents, these include the psoralen derivatives,including furocoumarins, the benzodipyrone derivatives, and thebis-azide derivatives. None of these molecules, however, work on thebasis of ionic activation. These reagents, especially the psoralenderivatives, are toxic, causing severe burning of the organism afterexposure to sunlight. Finally, the covalent crosslinks formed bypsoralens are not permanent, rather, they are degraded by UVirradiation.

Two recent articles reported the use or psoralen crosslinks of DNAsubstrates, the first by Van Houten et al., in "Action Mechanism of ABCExcision Nuclease on a DNA Substrate Containing a Psoralen Crosslink ata Defined Position", Proc. Natl Acad. Sci. U.S.A., 83, 8077-8081 (1986),and the second by Lee et al., in "Interaction of Psoralen-DerivatizedOligodeoxyribonucleoside Methyl-Phosphonates with Single-Stranded DNA",Biochemistry, 27, 3197-3203 (1988). Both articles reported covalentcross-linking between the DNA molecule and a complementary oligomer thatcontains a psoralen derivative. The covalent binding of the psoralenderivative to the DNA molecule was activated by UV irradiation.Accordingly, just like the Yakusaki patent application, the covalentcrosslinks formed by psoralens are not permanent, being degraded by UVirradiation.

The techniques of Northern and Southern blotting are two of the mostpowerful and frequently used procedures in molecular biology, see Wallet al., "Northern and Southern Blots", Methods Enz., 152, 572-573(1987). Yet the necessary manipulations are time consuming and are notlikely to be automated under current technology. Often thepolynucleotide (RNA, DNA) under analysis must first be fractionated bysize, transferred onto a solid support and then treated through a seriesof steps to ensure only specific binding of a probe. Detection of thehybridized products usually depends on radiolabelling, heavy metalderivatization or antibody complexation. The methods of blotting havebeen a staple of basic research, and now also serve in an everincreasing number of commercial kits used to diagnose genetic,malignant, and infectious diseases. See Landegren et al., "DNADiagnostics-Molecular Techniques and Automation", Science, 242, 229(1988). Related advances have also allowed these processes to aid inforensic science, see Higuchi et al., "DNA Typing from Single Hairs",Nature, 332, 543-546 (1988); and the Human Genome Project, see Conner etal., "Detection of Sickle Cell β'-Globin Allele by Hybridization withSynthetic Oligonucleotides", Proc. Natl. Acad. Sci. U.S.A., 80, 278-282(1983).

Psoralens have been used to randomly crosslink duplex DNA duringhybridization in order to facilitate Southern Blotting procedures. Thisnew test is referred to as Reverse Southern blotting. For example, seeGamper et al., "Reverse Southern Hybridization", Nucl Acids Res., 14,9943 (1986). Other biochemical and reduction activated reagents areneeded to replace or complement psoralens for sequence detection and toprovide an alternate set of conditions for duplex stabilization.

Accordingly, none of the related art describes or suggests using ionicactivation with aromatic silyloxy alkylating agents in order topermanently alkylate a biological molecule such as DNA.

Therefore, it is a purpose of the present invention to provide a newclass of ionically activated alkylating probes which form a permanentcovalent crosslink.

Another purpose of the present invention is to provide an ionicallyactivated alkylating probe which can be used in vivo.

A further goal of the present invention is to provide a new class ofionically activated Reverse Southern blotting reagents for conjugatingand permanently crosslinking target oligonucleotides and facilitateblotting procedures, sequence detection and nucleic acid fragmentation.

SUMMARY OF THE INVENTION

These and other purposes and goals are achieved by the present inventionwhich provides a process and alkylating agent for selectively andpermanently alkylating a target molecule. The process includes a step ofproviding an alkylating agent, namely a silyloxy aromatic derivative,capable of alkylating a target molecule. The silyloxy aromatic compoundmay non-specifically localize to the target molecule. Alternatively, theprocess may further include linking the silyloxy aromatic derivative toa probe capable of localizing it to the target molecule. The probe, suchas an oligonucleotide, may be capable of recognizing a predeterminedbinding site on a target molecule, such as a specific sequence of anucleic acid, which is complementary to the probe.

The preferred silyloxy aromatic compound of the invention comprises asubstituted aromatic ring system, to which have been attached a silyloxymoiety (--OSiR₆ R₇ R₈) and, in conjugation with the silyloxy moietythrough the ring system, at least one substituted methyl group (--CR₉R₁₀ X). In the preferred embodiment R₆, R₇ and R₈ are alkyl or aromaticgroups. Most preferably R₆ and R₇ are methyl groups while R₈ is at-butyl moiety. R₉ and R₁₀ are H or an alkyl or aromatic group. If morethan one --CR₉ R₁₀ X group occupies a position on the ring system and inconjugation with the silyloxy moiety then the --CR₉ R₁₀ X moieties neednot be identical, with R₉, R₁₀, and X being selected independently ofthe R₉, R₁₀ and X of other --CR₉ R₁₀ X moieties.

The siloxy aromatic derivatives of the present invention contain atleast one substituted aromatic center, such as a substituted benzenering. If more than one aromatic ring is included, the rings arepreferably fused, such as a naphthalene, anthracene, or phenanthrene.One or more rings may optionally be heterocyclic, containing a ringmember other than carbon, such as oxygen or nitrogen, while preservingthe aromatic character of the ring. Suitable heterocyclic aromaticsystems include, for example, quinolines, azanthracenes, andazaphenanthrenes.

The alkylating agent may have the general formula: ##STR1## When R₁=--OSiR₆ R₇ R₈, then R₂ and/or R₄ can be =--CR₉ R₁₀ X.

When R₂ =--OSiR₆ R₇ R₈, then R₁, R₃ and/or R₅ can be =--CR₉ R₁₀ X.

When R₃ =--OSiR₆ R₇ R₈, then R₂ and/or R₄ can be =--CR₉ R₁₀ X.

When R₄ =--OSiR₆ R₇ R₈, then by symmetry R₅ is equivalent to R₂ asdescribed above.

When R₅ =--OSiR₆ R₇ R₈, then by symmetry R₅ is equivalent to R asdescribed above.

R₆, R₇, R₈ =various alkyl or aromatic groups;

X=leaving group, and

wherein R₉ and R₁₀ can be H, or an organic derivative, such as analiphatic or aromatic group.

The --CR₉ R₁₀ X group is positioned on any of the carbon atoms of thering structure in resonance with the oxygen of the silyloxy moiety; L isa linking group optionally present for attachment to a probe which maybe positioned at any carbon atom of the ring, and P is a probe forbinding to a target molecule. Preferably, the targeted alkylating agentof the present invention has the general formula: ##STR2## In thisembodiment, an --L--Pr moiety may optionally be present at any of theunoccupied positions of the benzene ring. R₉ and R₁₀ are independentlyH, or an organic derivative, such as an aliphatic or aromatic group, andX is a leaving group.

The alkylating agent need not be restricted to a single aromatic ring.For example it may have a multi-ring structure, ##STR3##

Alternatively, the alkylating agent may have a ring structure comprisingmore than one aromatic ring, including one or more heterocyclic aromaticrings, such as acridine, a well known intercalator: ##STR4##

The alkylating agent, optionally linked to a localizing probe, is thenintroduced into a system containing the target molecule to allow thealkylating agent and/or the probe to associate, i.e., hybridize, withthe target molecule and to thereby localize the silyloxy aromaticderivative near the target molecule. The targeted alkylating agent isactivated by ionic strength, which causes covalent bonding between thesilyloxy aromatic derivative and the target molecule. If a probe isused, the covalent bonding occurs at a site proximal to the associationsite of the probe.

In a preferred embodiment, the X group is a displaceable reactive moietyattached to an alkyl group positioned on a carbon atom of the silyloxyaromatic ring. Examples of such groups include Br, Cl, F, I, --OAc,--OH, --OSO₂ CH₃, --OSO₂ C₆ H₄ CH₃ --p, --OCH₂ CH₃, --OCONHCH₃,--OCONHCH₂ CH₂ R, --OC₆ H₄ NO₂, --OC₆ H₅, and --SC.sub. H₅. Thealkylating agent is activated, in vitro by adjusting ionic strength, andin vivo by naturally occurring ionic strength. The ionic strength of thein vitro environment capable of activating the alkylating agent isbetween about 1 mM and about 10M, preferably between about 100 mM andabout 2M.

For a better understanding of the present invention reference is made tothe following description made in conjunction with the figures, thescope of which is defined in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates ionic activation (M⁺ X⁻) of a conjugated target (T)and probe (Pr) linked to a preferred silyloxy aromatic molecule tocreate the reactive intermediate of the present invention generated foralkylation of the target DNA.

FIG. 2 is an autoradiogram of a denaturing polyacrylamide gelelectrophoresis showing the ionic activated cross-linking reactionbetween a probe and a DNA target in accordance with the presentinvention, as described in Example 2, Protocol 1.3.

FIG. 3 is an autoradiogram showing that fluoride is not the onlypossible ionic triggering agent, as described in Example 2.

FIG. 4 illustrates proton NMR data concerning the fluoride-mediateddeprotection of a silyloxy aromatic compound of the present invention(starting material=SM), as described in Example 8.

FIG. 5 illustrates proton NMR data concerning production ofpara-nitrophenol as an indicator of hydrolysis of a deprotected silyloxyaromatic compound of the present invention, as discussed in Example 8.

FIG. 6 illustrates spectrophotometric data showing that a large excessof fluoride does not influence the second stage (hydrolysis) ofdeprotection of a silyloxy aromatic compound of the invention, asdescribed in Example 9.

FIG. 7 illustrates spectrophotometric data showing that the second stageof solvolysis of a silyloxy aromatic compound of the invention is notmediated by non-fluoride salts, as described in Example 9.

FIG. 8 illustrates spectrophotometric data showing that a nucleoside,deoxyguanine (dG), in either the presence or absence of LiClO₄, does notpromote solvolysis of a silyloxy aromatic compound of the invention, asdescribed in Example 9.

FIG. 9 shows an autoradiogram of denaturing polyacrylamide gel (20%)used to identify the cross-link product of duplex DNA byt-butyldimethyl-2,5-dibromomethylenephenol ether (Compound 11.2) asdescribed in Example 11.

DETAILED DESCRIPTION OF THE INVENTION

DNA alkylation has become a mainstay of cancer chemotherapy despite theoften devastating side effects of the prescribed drugs. One of theextreme examples is the nitrogen mustard mechlorethamine which must beadministered by intravenous injection to prevent severe reaction withlocally exposed tissue. Gilman et al., "The Pharmacological Basis ofTherapeutics", 6th ed Macmillan, New York (1980), Ch. 55. This compoundis so reactive that it will decompose within minutes by reacting withcellular constituents and even water. A more controlled reaction may beachieved through the use of compounds requiring cellular activation as aprerequisite to DNA alkylation. In this manner, the undesirable sidereactions of an active agent may be minimized prior to its entry into acell. For example, cylcophosphamide must be oxidatively metabolizedbefore DNA modification may occur and consequently, this drug exhibits amuch lower acute toxicity. Similarly, mitomycin requires a reductiveconversion before it can derivatize and cross-link nucleic acids.

The consummate alkylating agent for chemotherapy has yet to bedeveloped. All currently prescribed alkylating agents suffer in partfrom unavoidable side reactions with non-target components of a cell ororganism. Despite the use of various strategies to minimize thedeleterious effects of these agents, none has overcome the necessity ofgenerating a reactive species at some distance from the intended site ofmodification. Even the most selective drugs that require metabolicactivation cannot overcome problems associated with the necessarydiffusion of a highly reactive species to a cell nucleus and finally toits chromosomes. During this time, all nucleophilic components, not justDNA, are subject to modification. A superior compound would instead havethe ability to diffuse to DNA in a latent form and become reactive onlywhen bound within the structure of DNA. We have developed silyloxyaromatic derivatives as latent alkylating agents for in vitro DNA probetechnologies. Removal of the silyl group and unmasking of the reactiveagent was predicted and confirmed to proceed with addition of fluorideion. However, the local environment established by duplex DNA alsopromotes the spontaneous solvolysis and elimination of the silyl groupin the absence of fluoride. This exceptional result is only detected inthe presence of duplex DNA. Single strands do not have the capacity toactivate the silyl derivative. Therefore, the latent appendage istransformed into a powerful alkylating agent solely by its associationwith duplex DNA.

The invention further contemplates the use of silyloxy aromaticcompounds as non-specific DNA alkylating agents. If non-specificreaction is desired, as it is in most types of chemotherapeutics, then aprobe that localizes the silyloxy aromatic compound next to DNA, withlimited or no regard for any specific DNA sequence, is sufficient. Twonon-limiting examples of such probes include distamycin, a compound thatbinds relatively non-specifically to the minor groove of duplex DNA, andacridine, an intercalator that inserts non-specifically into the doublehelical structure of duplex DNA.

Alternatively, the linker-probe moiety could be dispensed with entirely,in situations where non-specific reaction is desired, if the silyloxycompound itself has appreciable affinity for duplex DNA. Such silyloxycompounds would include, for example, fused and planar ring systems of 3or more rings, such as anthracenes and phenanthrenes.

Upon analysis of the chemical basis of salt-induced DNA alkylation,other related silyloxy aromatic compounds in accordance with the presentinvention have been identified. These compounds are "latent" alkylatingagents, i.e., they are essentially unreactive except when activated withsalts and in the presence of duplex DNA.

At least two independent mechanisms of activation of these compoundshave been observed. A fluoride-dependent mechanism occurs in the mannerdescribed elsewhere herein. This is a very efficient and apparentlyuniversally applicable method for promoting DNA alkylation. Dataindicate that this mechanism induces alkylation by model silyloxyaromatic reagents, by silyloxy aromatic-linker-probe complexes, and bythe complexes hybridized to target DNA.

An alternative mechanism involves the mediation of alkylation by theionic strength of the environment. Ionic strength alone, in the absenceof fluoride, can induce alkylation of duplex DNA. However, thismechanism is also less efficient than the fluoride-dependent mechanism,requiring more time to produce an equivalent degree of alkylation. Theionic strength-dependent mechanism also lacks the capacity to activate asilyloxy aromatic model compound in the absence of fluoride or duplexDNA. The ionic strength-dependent model also fails to alkylatesingle-stranded DNA in the absence of fluoride, while thefluoride-dependent mechanism alkylates single and double stranded DNAwith substantially equal efficiency.

The ionic strength-dependent mechanism is independent of the nature ofthe salt (with the exception of fluoride). For example LiClO₄, achaotropic agent, has been found to be as effective in inducingalkylation of duplex DNA as NaCl, despite the contrasting natures of thesalts. NaCl is normally associated with increased hydrophobicinteractions while LiClO₄ is normally associated with decreasedhydrophobic interactions. Therefore, general ionic strength, nothydrophobic effect, appears to be critical to the ionicstrength-dependent mechanism of DNA alkylation by the silyloxy aromaticcompounds of the present invention.

In accordance with a preferred embodiment of the present invention, anaromatic derivative is conjugated to a probe which has potential forselective alkylation of target biological molecules. It is believed thatthe conjugated aromatic derivatives will not react indiscriminately withbiological materials other than the target molecules.

The preferred silyloxy aromatic compound of the invention comprises asubstituted aromatic ring system, to which have been attached a silyloxymoiety (--OSiR₆ R₇ R₈) and at least one substituted methyl group (--CR₉R₁₀ X) in conjugation with the silyloxy moiety through the ring system,i.e., at any carbon in the ring system that participates in resonancewith the oxygen of the silyloxy moiety. In the preferred embodiment R₆,R₇ and R₈ are alkyl or aromatic groups, most preferably R₆, R₇ aremethyl groups while R₈ is a t-butyl moiety. R₉ and R₁₀ are H or an alkylor aromatic group. If more than one --CR₉ R₁₀ X group occupies aposition on the ring system in conjugation with the silyloxy moiety,then the --CR₉ R₁₀ X moieties need not be identical, with R₉, R₁₀ and Xbeing selected independently of the R₉, R₁₀ and X of other --CR₉ R₁₀moieties.

The silyloxy aromatic derivatives of the present invention contain atleast one substituted aromatic center, such as a substituted benzenering. If more than one aromatic ring is included, the rings arepreferably fused, such as naphthalenes, anthracenes, and phenanthrenes.One or more rings may optionally be heterocyclic, containing a ringmember other than carbon, such as oxygen or nitrogen, while preservingthe aromatic character of the ring suitable heterocyclic aromaticssystems include, for example, quinolines, azanthracenes, andazaphenanthrenes.

A novel aromatic alkylating probe composition may have the followinggeneralized formula: ##STR5## When R₁ =--OSiR₆ R₇ R₈, then R₂ and/or R₄can be =--CR₉ R₁₀ X.

When R₂ =--OSiR₆ R₇ R₈, then R₁, R₃ and/or R₅ can be =--CR₉ R₁₀ X.

When R₃ =--OSiR₆ R₇ R₈, then R₂ and/or R₄ can be =--CR₉ R₁₀ X.

When R₄ =--OSiR₆ R₇ R₈, then by symmetry R₄ is equivalent to R asdescribed above.

When R₅ =--OSiR₆ R₇ R₈, then by symmetry R₅ is equivalent to R asdescribed above.

R₆, R₇, R₈ =various alkyl or aromatic groups;

X=leaving group, and

Wherein R₉ and R10 can be H, or an organic derivative, such as analiphatic group or an alkyl group.

In which CR₉ R₁₀ X is positioned on any of the carbon atoms of the ringstructure; L is a linking group for attachment to a probe which may bepositioned at any carbon atom of the ring, and Pr is a probe for bindingto a target molecule. Preferably, the targeted alkylating agent of thepresent invention has the general formula: ##STR6## The alkylating agentneed not be restricted to a single aromatic ring. For example, the agentmay have a fused multi-ring structure, such as a substitutednaphthalene: ##STR7## or, a substituted anthracene: ##STR8##

Alternatively, the alkylating agent may have a ring structure comprisingmore than one aromatic ring, including one or more heterocyclic aromaticrings, such as acridine, a well known intercalator: ##STR9##

The targeted alkylating agent, optionally linked to a localizing probe,is then introduced into a system containing the target molecule to allowthe alkylating agent and/or the probe to associate, i.e. hybridize, withthe target molecule and to thereby localize the silyloxy aromaticderivative near the target molecule. The targeted alkylating agent isactivated by ionic strength, which causes covalent bonding between thesilyloxy aromatic derivative and the target molecule. If a probe isused, the covalent bonding occurs at a site proximal to the associationsite of the probe.

In these embodiments, the silyloxy aromatic probe alkylates a targetmolecule after activation by an ionic signal. In these embodiments, X isa leaving group connected to an alkyl chain positioned on an aromaticring structure. The alkyl chain is connected at one end to the aromaticring and includes R₉, an organic derivative.

Thus X may include a leaving group, such as Cl, Br, F, I, --OCOR, --OH,--OSO₂ CH₃, --OSO₂ C₆ H₄ CH₃ --p, --OR, --OCONHR, --OCONHCH₂ CH₂ R,--OC₆ H₄ NO₂ (nitrophenol), --OC₆ H₅ (phenol), and --SC₆ H₅(thiophenol).

In all of these compositions, the linking group L is made up of a --R₁₀--R₁₁ --R₁₂ --. Generally, the R₁₀ group may include a group for linkingto the silyloxy aromatic derivative including NH, S, O or CH.sub.. TheR₁₁ group can include any spacer group which can link R₁₀ and R₁₂, suchas an alkyl chain. The R₁₂ group is any group which can link to amodified oligonucleotide or other probe Pr, examples of these are --NH₂,--SH, --OH and --COOH. The probe Pr includes any localizing moiety, suchas an oligonucleotide, protein, intercalator, or other molecule thatpreferentially localizes to an organic molecule, including DNA, RNA, orprotein. The oligonucleotide, whether DNA or RNA may be linked to R₁₂ ateither its 5' or 3' terminus.

Alternatively, the oligonucleotide may be linked to R₁₂ at anyoligonucleotide base, or phosphoribose backbone suitably modified inaccordance with methods well known to those persons skilled in the art.Examples include methods described by the following publications:

1. Brakel, Ed., "Discoveries in Antisense Nucleic Acids", GulfPublishing Company, Houston (1989).

2. Englisch et al., "Chemically Modified Oligonucleotides as Probes andInhibitors", Angewandte Chemie Int. Ed., 30, 613-722 (1991).

3. Nielsen, "Sequence-Selective DNA Recognition by Synthetic Ligands",Bioconjugate Chemistry, 2, 1-12 (1991).

4. Uhlmann et al., "Antisense Oligonucleotides: A New TherapeuticPrinciple", Chemical Reviews, 90, 543-584 (1990).

5. Goodchild, "Conjugates of Oligonucleotides and ModifiedOligonucleotides: A Review of Their Synthesis and Properties",Bioconjugate Chemistry, 1, 165-187 (1990).

6. Gebeyehu et al., "Novel Biotinylated Nucleotide-Analogs for Labellingand Colorimetric Detection of DNA", Nucl. Acids Res., 15, 4513-4534(1987).

7. Jager et al., "Oligonucleotide N-Alkyl-phosphotamides: Synthesis andBinding to Polynucleotides", Biochemistry, 27, 7237-7246 (1988).

8. Cocuzza, "Total Synthesis of 7-Iodo-2,', 3'-Dideoxy-7-DeazapurineNucleosides, Key intermediates in the Preparation of Reagents for theAutomated Sequencing of DNA", Tet. Lett., 29, 4061-4064 (1988).

9. Hanna et al., "Synthesis and Characterization of5-[(4-Azidophenacyl)thio]uridine 5═-Triphosphate, a CleavablePhoto-Cross-Linking Nucleotide Analogue", Biochemistry, 28, 5814-5820(1989).

10. Gibson et al., "Synthesis and Application of DerivatizableOligonucleotides", Nucl. Acids Res., 15, 6455-6467 (1987).

11. Nelson et al., "A New and Versatile Reagent for IncorporatingMultiple Primary Aliphatic Amines Into Synthetic Oligonucleotides",Nucl. Acid Res, 17, 7179-7186, (1989).

In a preferred embodiment of the invention, described in Examples 1 and2, the silyloxy aromatic alkylating probe is activated by an ionicsignal. For in vitro use the preferred ionic signals are KF, NaF, CsFand other salts (MX), defined as salts of a metal (M) and an anion (X).These, however, are not the only possible ionic triggering agents.Rather, the triggering signal is dependent on a general increase inionic strength. Accordingly, silyl-containing reactive centers, such asSi:R₆ R₇ R₈, as defined above, can be used for both in vitro and in vivouses.

A preferred embodiment of the present invention has the followingstructure: ##STR10## Another embodiment (7) which was attemptedincludes: ##STR11## This embodiment, however, proved too reactive.

Another embodiment (8) was attempted, but it did not couple well to theprobe: ##STR12##

Another embodiment (9 ) was too unreactive, as it would only work innon-aqueous systems: ##STR13##

The present invention also describes a process for selectivelyalkylating a target molecule. A great number of useful clinical andlaboratory applications for which this process may be applied aredescribed for somewhat related processes in PCT published ApplicationNo. WO 85/02628 to Yabusaki et al., the disclosure of which isincorporated by reference herein. Also the process of Reverse SouthernBlotting is described generally in the Background of the Related Art,supra.

Generally, the process of this invention may be carried out by firstproviding a probe for recognizing a predetermined binding site on atarget molecule. The probe may include a strand of DNA, RNA, or aprotein. See, for example, Praseuth et al., "Sequence-Specific Bindingand Photocrosslinking of α and βOligodeoxynucleotides to the MajorGroove of DNA via Triple-Helix Formation", Proc. Natl. Acad. Sci.U.S.A., 85, 1349-1353 (1988). Alternatively it may include any othermolecule which can localize the probe to a target molecule. For example,Dervan, "Design of Sequence-Specific DNA-Binding Molecules", Science232, 464-471 (1986), describes a variety of natural and syntheticcompounds that bind DNA in a sequence-specific manner and which comprisea series of subunits linked by peptide bonds. Among these compoundspotentially useful as probes in accordance with the invention isactinomycin, which acts as an intercalator by means of a phenoxazonemoiety, and binds 4 base pairs above and below the intercalation site bymeans of identical cyclic pentapeptide lactones. Actinomycin exhibits apreference for 5'-NGCN-3' sequences. The Dervan article also describesan alternative mode of peptide binding to double-helical DNA, such asthe minor groove sequence specific binding exhibited by distamycin, acrescent-shaped tripeptide containing three N-methylpyrrole carboxamideslinked by peptide bonds. In addition, Dervan describes comparablesequence-selective binding of oligopeptides having up to 7 amide groups.Dervan also describes synthetic sequence specific probes utilizing acombination of intercalation and minor groove binding as the means foridentifying double-helical DNA sequences up to 10 base pairs in length.The Dervan article illustrates that it is well known in the art that aprobe useful for the invention may optionally be an intercalatingmoiety, a peptide of variable length and structure or even a combinationof the two sorts of localizing molecule.

The Dervan publication also mentions the binding of organometalcomplexes to DNA. This type of localizing molecule is further describedin a review by Barton entitled "Metals and DNA: Molecular Left-HandedComplements", Science, 233, 727-734 (1986). The Barton publicationdescribes chiral metal complexes capable of various structuralinteractions with DNA including stereoselective intercalation, groovebinding, and direct coordination. Such metal complexes may be useful asprobes in accordance with the present invention.

The process carried out by providing a silyloxy aromatic derivative,which may be modified for linking to the probe molecule if desired. Theprobe is then linked to the silyloxy aromatic derivative to create atargeted alkylating agent. The target alkylating agent is introducedinto a system containing a target molecule, and the probe associateswith the target molecule localizing the linked silyloxy aromaticderivative near the target molecule. As illustrated in FIG. 1,crosslinking or covalent bonding is then initiated by activating thetargeted alkylating agent by an ionic signal, such as KF, other saltsMX, or the ionic signal can be the naturally occurring high ionicstrength region localized around polyanionic nucleic acids. A covalentbond is then formed between the aromatic derivative and the target,proximal to the association site of the probe with the target molecule.

In one preferred embodiment, the linking step includes a step ofadapting the silyloxy aromatic derivative by the addition of an acidiclinking group which is capable of being modified for linking to theprobe molecule. Preferably, the silyloxy aromatic derivative includes atleast one arm, --CR₉ R₁₀ X, attached to the aromatic ring, and inconjugation through the ring with the silyloxy moiety.

Ideally, drugs targeted at nucleic acid should be able to modify DNA orRNA sequences specifically and efficiently. Specific modification oftarget sequences can be achieved by using "antisense" or "triplexforming" techniques in which oligonucleotides are used to interfere withDNA or RNA functions. Alternatively, probes may be used which targetnucleic acids but which are not directed to specific sequences.

However, a number of factors are known to impede such techniques. Amongthese factors is that the exogenous oligonucleotide must travel to thetarget cell, must traverse the cell membrane, must find its target RNAor DNA in the cytoplasm or nucleus, must bind with high affinity andspecificity, and must exert the desired biological effect for thedesired period of time. To improve cellular uptake, stability, andaffinity, an agent can be formed by conjugating a functional group withan oligonucleotide. Optimally, the functional group would have activitywhich is induced solely by the target, i.e., only the target DNA or RNAwill trigger the reaction between the agent and the target itself.

Accordingly, in another embodiment of the invention, an oligonucleotideconjugate of a silyl phenol ether which is a latent o-quinone methideprecursor has been developed apparently possessing this targetedinducible activity. This conjugate has the following structure:##STR14##

This conjugate was developed as a latent alkylating agent for use as anin vitro DNA probe. The DNA target modification was induced by fluorideion, which was believed to promote the o-quinone methide intermediateformation. The desylilation apparently proceeds as follows: ##STR15##

DNA alkylation was also observed to take place in the presence ofnon-fluoride salts such as KCl, KBr, KClO₄, NaCl, NaBr, NaClO₄, LiClO₄,MgCl₂, and potassium phosphate. This list of salts is illustrative butnot exhaustive of the salts which are useful for the invention. In thissituation the alkylation and cross-linking of the target strand of DNAwith the conjugate may have a different mechanism from that reactioninduced by the fluoride salt.

A model compound, the synthesis of which is described in Example 7, wasdesigned to study the mechanism of fluoride induced DNA alkylation. Inparticular, the reactivity of the functional group moiety tonucleophiles such as DNA bases or the solvent are capable of beingstudied using this model compound. The model compound was synthesizedand its solvolysis in the presence of fluoride salts, non-fluoridesalts, and nucleoside was studied kinetically by ¹ H NMR (Example 8) andUV-VIS spectroscopy (Example 9).

Results of these analyses showed that only fluoride ion is capable ofdeprotecting the phenol hydroxy group and further promoting solvolysisof the model compound. It was also observed that non-fluoride salts anda nucleoside had no effect on solvolysis of the model compound.

These results were consistent with in vitro experimental results whichshowed that alkylation and cross-linking of target DNA with conjugateoccurs in the presence of non-fluoride salts only when the silyloxyaromatic compound associates with duplex DNA or when the target has beenpreviously hybridized with the conjugate. See Examples 1-5. It ispossible that a unique conformational microenvironment is formed whichrequires the hybridization of the target DNA with the conjugate with theaid of salts. It is believed that this microenvironment facilitates thegeneration of the quinone methide for the nucleophilic reaction betweenthe DNA bases and the functional group of the conjugate to produce thecross-linked product.

If in vivo use is desired, then suitably modified probes capable oftraversing cell membranes may be prepared, as well known to thoseskilled in the art, for example, as described by Blake et al.,"Hybridization Arrest of Globin Synthesis in Rabbit Reticulocyte Lysatesand Cells by Oligodeoxyribonucleoside Methylphosphonates", Biochemistry,24, 6139-6145 (1985); and by Agrawal et al., "OligodeoxynucleosidePhosphoramidates and Phosphorothioates as Inhibitors of HumanImmunodeficiency Virus", Proc. Nat'l. Acad. Sci. U.S.A., 85, 7079-7083(1988). These probes are then attached to the activated esters.

The following Examples further illustrate the various features of theinvention, and are not intended in any way to limit the scope of theinvention, which is defined in the appended claims.

In these Examples, we have shown that a preferred probe, as describedabove, causes selective alkylation of a DNA target, which has beenionically activated after the probe has hybridized with the target DNAsequence.

EXAMPLE 1 Preparation of Silyloxy Aromatic Ionically InducibleAlkylating Linked Probe

An ionic induced silyloxy aromatic alkylating linked probe was preparedin accordance with the invention. The probe was tested in vitro using asynthetic DNA target strand. The steps followed in the synthesis of arepresentative silyloxy aromatic ion-induced alkylating probe forcoupling to the 5' terminus of an oligonucleotide are generally shown inScheme A. ##STR16##

A parallel series of steps followed in the synthesis of a representativesilyloxy aromatic ion induced alkylating probe for coupling to the 3'terminus of an oligonucleotide are shown in Scheme B. ##STR17##

EXAMPLE 2 Preparation of the preferred silyloxy aromatic system suitablefor coupling to a probe-linker species (L-Pr).

The following general Scheme (Scheme 1) illustrates the steps taken inthe synthesis of preferred silyloxy aromatic molecules which aresuitable for coupling to probe (Pr)--linker (L) species, and directingthe ionically inducible covalent crosslinking system to a desired target(T). The method described in Scheme 1 is general enough for thepreparation of a number of useful derivatives of Compound 1.7. ##STR18##

Protocol 1.1: Synthesis of the Silyl Protected 1,2,5-TrisubstitutedPhenol Materials and Methods for the Synthetic Procedures

Proton magnetic resonance (¹ H NMR) spectra were recorded on a QE-300spectrometer. Chloroform-d was used as a solvent and TMS as reference.UV/VIS spectra were measured with a Perkin-Elmer Lambda 5spectrophotometer, and mass spectra were obtained with a HP 598A massspectrometer. Flash chromatography is a commonly used purificationtechnique described by Still et al., J. Org.. Chem., 43, 2923-2925(1978), and 230-400 mesh silica gel was used. Thin-layer chromatography(TLC) analysis utilized Machery-Nagel polygram Sil G/UV silica gelplates. Tetrahydrofuran (THF) was distilled from sodium, andacetonitrile was distilled from CaH₂ immediately prior to use. Dimethylformamide (DMF), CCl₄ and triethylamine were stored over Linde 4-Amolecular sieves at least two days prior to use. Other materialsidentified in these Examples were obtained from commercial suppliers andused without further purification.

3-t-Butyldimethylsiloxyl-4-methylbenzoic acid (Compound 1.2)

t-Butyldimethylsilyl chloride (3.32 g, 22.1 mmol) was added to asolution of Compound 1.1 (1.12 g, 7.4 nmol) and triethylamine (1.79 g,16.2 mmol) in 30 mL THF. The mixture was heated at 40° C. overnight.After the reaction mixture was allowed to cool to room temperature, thetriethylammonium chloride was filtered out. The filtrate was thendiluted with ether (50 mL) and a few drops of distilled water was addedand stirred at room temperature for five hours. After evaporation of thesolvent, the product was purified by flash silica chromatography (ethylacetate:hexanes=1:3) to yield 1.60 g (81.5%) of a white solid: ¹ H NMR δ7.49 (s, 1H), 7.39 (s, 1H), 7.11 (d, 1H), 2.20 (s, 3H), 0.96 (s, 9H),0.31 (s, 6H).

3-t-Butyldimethylsiloxyl-4-methylbenzoic acid N-hydroxysuccinimide ester(Compound 1.3)

Dicyclohexylcarbodiimide (DCC, 1.17 g, 5.7 mmol) was added to a solutionof N-hydroxysuccinimide (0.28 g, 7.1 mmol) and Compound 1.2 (1.27 g, 4.8mmol) in DMF (50 mL) and stirred overnight at 4° C. This mixture wasthen diluted with ether (50 mL) and water (50 mL), filtered andconcentrated. The remaining residue was purified by flash silicachromatography (ethyl acetate:hexanes=1:3) to yield 1.13 g (65.3%) of awhite solid: ¹ H NMR δ 7.60 (d, 1H), 7.41 (s, 1H), 7.18 (d, 1S), 2.83(s, 4H), 2.23 (s, 3H), 0.96 (s, 9H), 0.18 (s, 6H). LRMS m/z 363 (M⁺),217, 189, 85.

N-13-t-Butyldimethylsiloxyl-4-methylbenzoyl) glycine (Compound 1.4)

An aqueous solution (50 mL) of glycine (0.11 g, 1.5 mmol) was combinedat room temperature with a solution of Compound 1.3 (0.44 g, 1.2 mmol)in acetonitrile (50 mL) and triethylamine (0.14 g, 1.2 mmol). Thismixture was manually shaken for two minutes and then washed with ether(50 mL). The aqueous phase was acidified to pH 2 with 6N HCl andextracted with ether (3×50 mL). The combined organic phases wereevaporated and the product was purified by flash silica chromatography(ethyl acetate:hexanes=1:1) to yield 0.31 g (74.6%) of a white solid: ¹H NMR δ 7.26 (m, 3H), 6.64 (m, 1H), 4.02 (d, 2H), 2.20 (s, 3H), 0.98 (s,9H), 0.22 (s,6S). LRMS m/z 323 (M⁺), 221, 149, 99.

N'-(3-t-Butyldimethylsiloxyl-4-methyl-benzozyl)glycine-N-hydroxysuccinimide ester (Compound 1.5)

The method described for the synthesis of Compound 1.3 was also used toproduce Compound 1.5 (62.2% yield). ¹ H NMR δ 7.24 (m, 3H), 6.50 (m,1H), 4.59 (d, 2H), 2.86 (s, 4H), 2.24 (s, 3H), 1.01 (s, 9H), 0.24(s,6H). LRMS m/z 420 (M⁺), 348, 190.

N'-(3-Butyldimethylsiloxyl-4-bromoethyl)benzoyl) glycineN-hydroxysuccinimide ester (Compound 1.6)

N-bromosuccinimide (NBS) (0.07 g, 0.4 mmol) was added to a solution ofCompound 1.5 (0.12 g, 0.29 mmol) in CCl₄ (10 mL). The mixture was thenmaintained at 20° C. and irradiated with a 275 W sunlamp (Sears,#34-7105) for fifteen minutes. After the solid succinimide was filteredaway, the filtrate was evaporated. The remaining residue was purified byflash silica chromatography (ethyl acetate:hexanes=1:3) to yield 0.08g(58.9%) of a white solid. ¹ H NMR δ 7.26 (m, 3H), 6.76 (m, 1H), 4.60 (d,2H), 4.50 (s, 2H), 2.86 (s, 4H), 1.01 (s, 9H), 0.31 (s, 6H). LRMS m/z344, 342, 263, 245.

N'-[3-t-Butyldimethylsiloxyl]-4-(p-nitrophenoxy) -benzoyl]glycineN-hydroxysuccinimide ester (Compound 1.7a)

Potassium p-nitrophenolate (0.02 g, 0.2 mmol) was added to a solution ofCompound 1.4 (0.08 g, 0.2 mmol) in freshly distilled acetonitrile (2mL). The mixture was stirred at room temperature for one hour and thenwater and ether (10 mL of each) were added. The aqueous phase was washedwith 3×10 mL of ether. The combined ether fractions were dried and theremaining residue was purified by flash silica chromatography to yield ayellowish solid (0.05 g, 59.4%). ¹ H NMR δ 8.32 (d, 2H), 7.60 (d, 2H),7.37 (m, 3H), 6.52 (m, 1H), 5.53 (d, 2H), 5.18 (s, 2H), 2.67 (s, 2H),1.02 (s, 9H), 0.30 (s, 6H). LRMS m/z 419, 349, 275, 189.

N'-[3-t-Butyldimethylsiloxyl-4-(phenoxymethyl) benzoyl]glycineN-hydroxysuccinimide ester (Compound 1.7b)

Potassium phenolate (0.01 g, 0.1 mmol) was added to a solution ofCompound 1.6 (0.02 g, 0.1 mmol) in freshly distilled acetonitrile (2mL). The mixture was stirred at room temperature for one hour and waterand ether (10 mL of each) were added. The aqueous phase was washed 3×10mL of ether. The combined ether fractions were dried and the remainingresidue was purified by flash silica chromatography to yield a whitesolid (0.01 g, 49%). ¹ H NMR δ 7.56 (d, 2H), 7.32 (m, 6H), 6.72 (m, 1H),5.17 (s, 2H), 4.60 (d, 2H), 2.66 (s, 4H), 1.01 (s, 9H), 0.29 (s, 6H).

N'-[3-t-Butyldimethylsiloxyl-4-(thiophenoxymethyl) benzoyl]glycineN-hydroxysuccinimide ester (Compound 1.7c)

This was synthesized under equivalent procedure as described forCompound 1.7a and Compound 1.7b, above, the adaptation of which is wellwithin the knowledge of those skilled in the art. ¹ H NMR δ 7.37 (m,8H), 6.60 (m, 1H), 4.52 (d, 2H), 4.04 (s, 2H), 2.82 (s, 4H), 0.95 (s,9H), 0.2 3 (s, 6H).

Protocol 1.2: Coupling the reactive centers (Compound 1.7) to a sequencedirecting oligonucleotide-linker (L-Pr) Materials and Methods forCoupling Procedures

Oligonucleotides were synthesized by standard solid phasephosphoramidite methods on a Dupont Coder 300 (Department ofPharmacology, SUNY at Stony Brook) and on a Biosearch instrument byClontech Laboratories, Inc. (Palo Alto, Calif.). When necessary, theoligonucleotides were also purified and deprotected by standardprocedures. Reverse phase (C-18) separation and analysis utilized aVarian 5000 HPLC controller, Varian 2050 variable wavelength detector,Hewlett Packard 3390A recording integrator and Spherex 5 μM C-18 column(Phenomenex). UV/VIS spectra were recorded on a Perkin Elmer Lambda-5spectrophotometer.

Preparation of the Oligonucleotide (Pr) Derivatized at the 5' End with aHexamethylamino Group (L-Pr)

The hexamethylamino linker was attached to the 5' end of the nascentoligonucleotide (Pr, ACGTCAGGTGGCACT) during the last step of the solidphase synthesis by using a monomethyoxytrityl protected hexamethylaminoprecursor (N-MMT-CG-AminoModifier supplied by Clontech Laboratories,Inc.). The protecting group was released after the complete synthesis bytreating the crude material with 80% acetic acid for 30 minutes. Thefree trityl derivative was removed by ether extraction and theoligonucleotide aminolinker derivative was stored as an aqueous solution(-200° C.) before coupling to the reactive centers.

Coupling the activated ester (Compound 1.7a) to the aminolinkeroligonucleotide probe (L-Pr)

A solution of 2 mg 1.7a in DMF (200 μL) was combined with a solution ofL-Pr (A₂₆₀ =3.0 absorbance units [AU]) in 0.25M3-(N-morpholino)-propanesulfonic acid at pH 7.5) (200 μL). This mixturewas left undisturbed at 40° C. for 5 hours. The coupled product,designated Compound 1.7a-L-Pr, was purified by reverse phase (C-18)chromatography using a gradient of 10% acetonitrile in 45 mMtriethylammonium acetate pH 6 to 30% acetonitrile in 30 mMtriethylammonium acetate pH 6 over 30 min (1 mL/min). The desiredmaterial eluted with a retention time of 23 min and, after collection,was immediately frozen and dried under high vacuum (20% yield based onrecovered A₂₆₀).

Protocol 1.3: Preparation of target (T) and Modification of T with thereagent Compound 1.7a-L-Pr.

The target strand (T, AGTGCCACCTGACGTCTAAG) was prepared in the samemanner as the oligonucleotide Pr described in Protocol 1.2. For productdetection, T was labeled with ³² P (*pT) in accordance with theprocedures described by Maniatis et al., in Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982).

Probe-target Crosslinking and Detection

The reaction between the probe (Compound 1.7a-L-Pr) and target sequences(T) was characterized in a standard reaction mixture (10 μL) containing1 mM potassium phosphate pH 7, 6 nM Compound 1.7a-L-Pr and 6 nM *pT (20nCi). Samples were incubated at 4° C. for no less than 10 min and thenan aqueous solution of KF was added to a final concentration of 10-250mM. This treatment activated the system for covalent crosslinking of thehybridized strands. This process was quenched after 10 min (4° C.) byaddition of excess DNA (for example, T) and placing samples on dry ice.The volume of each sample was then reduced by 50% under high vacuum and5 μL of 80% formamide was added in preparation for electrophoreticanalysis.

FIG. 2 shows an autoradiogram of a denaturing 20% polyacrylamide gelthat was used to demonstrate the successful application of Compound1.7a-L-Pr. *T is the target strand and D-silyl-pNP is Compound1.7a-L-Pr. The concentrations, quenching and analyses are all the sameas described above for FIG. 1. Reactions were carried out at roomtemperature for 30 minutes. Lane 1 indicates that no alkylation of thetarget (crosslinking) occurred in the absence of fluoride. Lane 2 is thepositive control, demonstrating that the crosslinking was triggered bythe presence of 100 mM fluoride. Lane 3 shows that the reagent can beneutralized by treatment with 100 mM fluoride (40° C., 30 minutes)before *T is added. Lane 4 proves that the oligonucleotide reagent isspecific for complementary sequences. A non-complementaryoligonucleotide (X) 14 nucleotides long ([³² P]-5'-CATGCGTTCCCGTG) didnot react with Compound 1.7a-L-Pr after addition of 100 mM fluoride. Forthe samples in lanes 5-9, the fluoride concentration was varied from0.0-250 mM.

FIG. 3 shows that fluoride is not the only possible triggering agent.The signal for inducing reaction is not so much dependent on fluoride asit is dependent on a general increase in ionic strength. Accordingly,silyl containing reactive centers can be used for both in vivo and invitro uses. No other ionic strength dependent covalent binding reagenthas ever before been proposed or tested.

Protocol 1.4: Synthesis of Reactive Centers with Reactivity Similar toCompounds 1.7a-c.

By treating Compound 1.6 with various nucleophiles (X), a series ofrelated appendages for triggered reaction were produced: ##STR19##

EXAMPLE 3

Reactive centers other than those represented by Compound 1.7 have beenconstructed for inducible and selective crosslinking of the complexformed by a probe (L-Pr) and target (T). ##STR20##

Protocol for the Preparation of a Reactive Center Designed for Couplingto an Aminolinker Probe (L-Pr) 3-(5-methylbenzoyl)propionic acid(Compound (2.2)

This compound was prepared by the method of Raval et al., J. Univ.Bombary, 7, Pt. 3, 184 (1983); CA 33, 3779 (1989). p-Cresol (4.0 g, 37mmol) and succinic anhydride (3.4 g, 34 mmol) were combined in1,1,2,2-tetrachloroethane (40 mL) and the mixture was heated to 60° C.Aluminum chloride (9.5 g, 71 mmol) was then added to the solution at arate of 2 g/20 min. Once this was complete the reaction was heated to135° C. for 30 min. After cooling, water and ether (30 mL of each) wereadded and the aqueous layer was extracted with 3×20 mL of ether. Thecombined organic fractions were dried and the remaining residue waspurified by flash silica chromatography to yield 2.2 g (28.5%). ¹ H NMRδ 7.62 (s, 1H), 7.30 (d, 1H), 6.85 (d, 1 H), 3.32 (t, 2H), 2.35 (t, 2H),2.22 (s, 1H).

3-(2-t-Butyldimethylsiloxyl-5-methylbenzoyl) propionic acid (Compound2.3)

t-Butyldimethylsilyl chloride (2.3 g, 18.6 mmol) was added to a solutionof Compound 2.2 (1.0 g, 18.6 mmol), triethylamine (0.9 g, 8.9 mmol) andTHF (15 mL) at room temperature. The reaction mixture was then stirredfor three hours at 40° C. The triethylammonium chloride was precipitatedand removed after addition of 10 mL ethyl acetate:hexanes (3:1). Thefiltrate was separated by flash silica chromatography to yield a yellowliquid (1.45 g). This material was consistent with a disilyl derivativeof Compound 2.2 and could be used directly to form the desired product.For example, an ether solution (20 mL) of this liquid (0.5 g, 1.1 mmol)was treated with two drops of water and stirred overnight at roomtemperature. After the solvent was removed, the product was purified onflash silica chromatography to yield a white solid (0.3 g, 81%). ¹ H NMRδ 7.30 (s, 1H), 7.02 (d, 1H), 6.66 (d, 1H), 3.22 (t, 2H), 2.26 (t, 2H),2.18 (s, 3H), 0.85 (s, 9H), 0.14 (s, 6 H).

3- (2-t-Butyldimethylsiloxyl-5-methylbenzoyl) propionic acidN-hydroxysuccinimide ester (Compound 2.4)

N-Hydroxysuccinimide (0.064 g, 0.31 mmol) was added to a solution ofCompound 2.3 (0.1 g, 0.31 mmol) in DMF (3 mL). After this mixture wascooled to 4° C., DCC (0.036 g, 0.31 mmol) was added. The reactionmixture was stirred for three hours at 4° C. and then filtered to removethe dicyclo-hexylurea. The filtrate was washed with water, dried andevaporated. The remaining residue was separated by flash silicachromatography to yield a white solid (0.096 g, 74%). ¹ H NMR δ 7.14 (s,1H), 7.11 (d, 1H), 6.73 (d, 1H), 3.37 (t, 2H), 2.93 (t, 2H), 2.77 (s,4H), 2.22 (s, 3H), 0.92 (s, 9H), 0.21 (s, 6H).

3-(2-t-Butyldimethylsiloxyl-5-(bromomethyl)benzoyl) propionic acidN-hydroxysuccinimide ester (Compound 2.5)

NBS (0.067 g, 0.37 mmol) and Compound 2.4 (0.13 g, 0.31 mmol) werecombined in CCl₄ (3 mL). This solution was then maintained at 20° C. andirradiated with a 275 W sunlamp (Sears, #34-7105) for fifteen minutes.After the solid succinimide was filtered away, the filtrate wasevaporated. The residue remaining was purified by flash silicachromatography to yield a yellow solid (0.072 g, 45%). ¹ H NMR δ 7.65(s, 1H), 7.36 (d, 1H), 6.78 (d, 1H), 4.39 (s, 2H), 3.37 (t, 2H), 2.98(t, 2H), 2.76 (s, 4H), 0.94 (s, 9H), 0.30 (s, 6H).

3-(2-t-Butyldimethylsiloxyl-5-(chloromethyl)benzoyl) propionic acidN-hydroxysuccinimide ester (Compound 2.6a)

Potassium chloride (0.68 g, 0.15 mmol) was added to a solution ofCompound 2.5 (0.05 g, 0.1 mmol) in acetonitrile (5 mL). The reactionmixture was stirred for two hours at 40° C. and then washed. The organicphase was dried, evaporated and separated by flash silica chromatographyto yield a white solid (0.034 g, 72%). ¹ H NMR δ 7.65 (s, 1H), 7.32 (d,1H), 6.81 (d, 1H), 4.48 (s, 2H), 3.36 (t, 2H), 2.96 (t, 2H), 2.77 (s,4H), 0.93 (s, 9H), 0.24 (s, 6H).

Each of these silyloxy aromatic alkylating agents can be substituted byreplacing the bromo with the other X groups, such as acetate,p-nitrophenolate and the like, as described in Example 2.

EXAMPLE 4

In a generalized embodiment, the silyloxy substituent may be in directconjugation with the --CHX-- group (for example, ortho or para whenattached to a phenyl ring) and an appendage for joining the aromaticsystem to the linker-probe (L-Pr) may be designed in any known manner.However, not all combinations were found to be appropriate due to theintrinsic reactivity of specific arrangement of functional groups.##STR21##

3-(4-methoxylbenzoyl)propionic acid (Compound 3.2)

A solution of p-anisole (4.32 g, 50 mmol) and succinic anhydride (4.14g, 40 mmol) were combined in 1,1,2,2-tetrachloroethane (10 mL) andnitrobenzene (40 mL) at 4° C. Aluminum chloride (24.56 g, 180 mmol) wasthen added gradually. The temperature was kept at 0°-5° C. and stirredovernight. Water was added and neutralized to quench the reaction. Theaqueous phase was separated and washed with ether then reacidified andwashed with ether again. The ether fractions were combined, dried andevaporated. The remaining residue was purified by flash silicachromatography to yield a white solid (7.55 g, 88%). ¹ H NMR (CDCl₃) δ7.98 (d, 3H), 6.88 (d, 2H), 3.76 (s, 3H), 3.30 (t, 2H), 2.76 (t, 2H).

3-(4-Hydroxylbenzoyl)propionic acid (Compound 3.3)

The methoxy derivative, Compound 3.2 (14.54 g, 70 mmol), was dissolvedin iodine free hydriodic acid (150 mL) and refluxed at 140° C. for fourhours. After the resulting brown solution was cooled to roomtemperature, water was added and the mixture was neutralized. Theaqueous phase was then washed with ether; the organic layers werecombined, decolorized, dried and evaporated. The remaining residue waspurified by flash silica chromatography to yield a white solid (11.93 g,88%). NMR (CDCl₃) δ 8.20 (d, 2H), 7.02 (d, 2H), 3.32 (t, 2H), 2.88 (d,2H).

3-(4-Butyldimethylsiloxyl)benzoyl)propanoic acid (Compound 3.4)

t-Butyldimethylsilyl chloride (0.63 g, 4 mmol) was added a solution ofCompound 3.3 (0.23 g, 1 mmol), triethylamine (0.21 g, 20 mmol) and THF(20 mL); this was kept stirred at room temperature for 3 hours. Solventwas then evaporated and the residue was dissolved in ether, washed withdilute HCl and then by saturated bicarbonate. The organic phase wasdried and evaporated to yield the disilyl derivative of 3.3. Thismaterial could be purified by flash silica chromatography (0.36 g, 78%)and stored, or it could be used immediately. The disilyl compound (0.36g, 4 mmol) was dissolved in 2-propanol (10 mL) and stirred overnight atroom temperature. The solvent was removed by evaporation and replacedwith ether. This mixture was then washed with water, dried, evaporatedand separated on flash silica chromatography to yield a white solid(0.23 g, 82%). ¹ H NMR (CDCl₃) δ 7.88 (d, 2H), 6.86 (d, 2H), 3.26 (t,2H), 2.76 (t, 2H), 0.98 (s, 9H), 0.86 (s, 6H).

4-(4-t-Butyldimethylsiloxyl)phenyl-4-hydroxybutyric acid (Compound 3.5)

A mixture of Compound 3.4 (0.55 g, 2 mmol), NaBH₄ (0.04 g, 1 mmol) andmethanol (5 mL) was heated to 50° C. After 10 hours, the resulting solidwas removed by filtration and the solution was evaporated to dryness.The remaining residue was purified on flash silica chromatography toyield a white solid (0.31 g, 56%). ¹ H NMR (CDCl₃) δ 7.17 (d, 2H), 6.76(d, 2H), 4.67 (t, 1H), 2.48 (t, 2H), 2.07 (m, 2H), 0.98 (s, 9H), 0.10(s, 6H).

4-(t-Butyldimethylsiloxyl)phenyl)-4-acetoxybutyric acid (Compound 3.6)(X=acetate)

Acetic anhydride (0.13 g, 1 mmol), triethylamine (0.13 g, 1.2 mmol) andCompound 3.5 (0.22 g, 0.6 mmol) were mixed in CHCl₃ for 5 hours at roomtemperature. The solution was then washed with sodium bicarbonate,dilute HCl and finally dried and evaporated. The remaining residue waspurified on flash silica chromatography to yield a solid (0.15 g, 61%).¹ H NMR (CDCl₃) δ 7.06 (d, 2H), 6.60 (d, 2H), 5.26 (m, 1H), 2.43 (m,3H), 2.05 (q, 2H), 0.86 (s, 9H), -0.12 (s, 6H).

Using related chemical techniques derivatives were made in which X=Br(Compound 3.7) internal cyclization prevented further development ofthis specific approach.

EXAMPLE 5

In order to produce an alkylating agent that is selectively generated inthe presence of an ionic strength modifying agent (MX), such aspotassium fluoride, the reactive appendage should include a silyloxygroup. Related derivatives containing a silyl substitution at a benzylposition are believed to be too stable for the applications describedfor this invention. Specifically, the characteristics of the --O--Si(R)₃bond, but not a--(R)₂ C--Si(R')₃, are optimal for the controlledalkylation of a target. For example: ##STR22##

These compounds were synthesized and characterized. Compound 4.5 wasfound to be too stable for use in an aqueous system.

EXAMPLE 6 Synthesis and Characterization ofN'-(3-t-[butyldimethyl-siloxy]-4-(p-nitrophenoxy)benzoyl)glycyl-N-hydroxy succinimide Ester

This example provides an alternative protocol for the synthesisdescribed above in Example 1. This Example describes a syntheticprotocol as well as characterizing data which further refine theprotocol and data provided in Example 1. The silyloxy aromatic compoundsynthesized and characterized as described in this Example, whenconnected with a sequence-directing oligonucleotide, is a latentsite-selective alkylating reagent of DNA. See Examples 1-5.

General Methods. Melting points were measured with a Thomas-HooverUnimelt apparatus and are uncorrected. The ¹ H NMR and ¹³ NMR spectrawere measured with a General Electric QE-300 spectrometer and thechemical shifts are relative to the deuteriated solvent signal; couplingconstants, J, are reported in Hz and refer to apparent peakmultiplicities and not true coupling constants. The IR spectra wererecorded on a Perkin-Elmer Model 1600 FT-IR spectrophotometer withsamples dissolved in CHCl₃ in a liquid cell. UV-VIS spectra wererecorded on a Perkin-Elmer Lambda 5 spectrophotometer.

Materials. Toluene was distilled over sodium/benzophenone under nitrogenprior to use. HPLC grade acetonitrile was distilled over calcium hydrideunder nitrogen prior to use. All other chemicals were purchased fromAldrich Chemical Co., Inc. ##STR23##

Commercially available 3-hydroxy-4-methyl-benzoic acid (Compound 6.1)was used as starting material. The phenolhydroxy group was protected byTBDMS. The chemical yield of the synthesis of Compound 6.2 was improvedby using imidazole as base and DMF as solvent. This method is analternative to the procedure described above in Example 1 which usedtrimethylamine as the base and THF as the solvent. Protection andactivation of the carboxyl group by N-hydroxy succinimide producedCompound 6.3. Light induced bromination of Compound 6.3 by NBS requireda single equivalent of NBS to complete the reaction and to avoiddibromination. Since the solubility of Compound 6.3 is not high in CCl₄,some of the product may be lost during filtration to remove thesuccinimide by-product. Better chemical yield is achieved bychromatographic separation of the reaction mixture without filtration.Chemical yield of the nucleophilic substitution reaction by potassiump-nitrophenolate to produce Compound 6.5 was low. This may be due to thecompeting attack of the nitrophenolate on the silyl group to desilylateCompound 6.3. Evidence for this was produced insofar ast-butyldimethylsilyl-p-nitrophenol ether was separated out as aby-product. The nitro-phenolate might also attack the activated carboxylgroup to form some very polar products that stayed at the original pointon TLC. The yield of this step may be further optimized. The subsequentsyntheses of Compounds 6.6 and 6.7 were straightforward. The reactionconditions may be further optimized to improve the yield of this step.

3-t-Butyldimethylsiloxyl-4-methyl benzoic acid (Compound 6.2)

3-hydroxy-4-methylbenzoic acid (Compound 6.1) (2.17 g, 14.2 mmol) wasdissolved in 30 mL DMF, and then imidazole (4.85 g, 71.0 mmol) andt-butyldimethyl-silyl chloride (6.44 g, 42.6 mmol) were addedsuccessively. The reaction mixture was stirred at room temperature (20°C.) for 48 hours. H₂ O (50 mL) was added into the reaction mixture andit was extracted by ether. The ether solution was then combined with 2mL H₂ O and one drop of 6N HCl and it was stirred for 48 hours. Thenthis reaction mixture was washed by H₂ O, concentrated and subjected toflash silica gel chromatography to yield the product Compound 9.2 (3.67g, y=96.7%) as a white solid: m.p. 134.5°-135° C.; ¹ H NMR (CDCl₃) δ0.26 (S,6H), 1.03 (s,9H), 2.27 (s,3H), 7.22 (d, J=7.8 Hz, 1H), 7.48 (d,J=1.4Hz, 1H), 7.61 (dd, J=1.5, 7.8 Hz, 1H); ¹³ C NMR (CDCl₃) δ -4.21,17.19, 18.29, 25.78, 119.64, 123.06, 127.99, 130.95, 135.92, 153.94,171.82; IR (CHCl₃) 3600-2600 (broad), 2957, 2930, 2859, 1691, 1607,1577, 1504, 1418, 1273 cm⁻¹ ; UV (CHCl₃) λ_(max) =293 nm; MS (EI) m/z(rel intensity) 266 (10.4, M⁺), 209 (100).

3-[(t-Butyldimethylsiloxyl)4-methylbenzoyl N-hydroxy-succinimide ester(Compound 6.3)

N-hydroxysuccinimide (1.94 g, 16.8 mmol) was added into a DMF (100 mL)solution of Compound 6.2 (3.00 g, 11.3 mmol ) and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (2.59 g, 13.5 mmol)at 0° C. The mixture was stirred for 20 hours at 4° C. and then it wasdiluted with brine and extracted with ether. The ether solution wasconcentrated and the residue was purified by flash silica gelchromatography (Hexane: ethyl acetate=3:1) to yield the product (3.71 g,y=90.8%) as a white solid: m.p. 103.5°-104° C.; ¹ H NMR (CDCl₃) δ 0.27(s,6H), 1.04 (s,9H), 2.31 (s,3H), 2.92 (s,4H), 7.28 (d, J=7.7 Hz, 1H),7.49 (d, J =1.4 Hz, 1H), 7.68 (dd,J=1.5, 7.8 Hz, 1H); .sup. 13 C NMR(CDCl₃) δ -4.25, 17.27, 18.23, 25.69, 25.85, 119.83, 123.37, 123.59,131.30, 137.52, 154.21, 161.82, 169.18; IR(CHCl₃) 3018, 2955, 2931,2858, 1768, 1741, 1605, 1576, 1503, 1413, 1288, 1210 cm⁻¹ ; UV (CHCl₃)λ_(max) =301 nm; MS(EI) m/z (rel intensity) 363 (5.0, M⁺), 306 (38.6),249 (59), 135 (100).

3-(t-Butyldimethylsiloxyl)-4-bromomethylbenzoyl N-hydroxysuccinimideester (Compound 6.4)

N-bromosuccinimide (1.46 g, 8.19 mmol) was added to a solution ofCompound 6.3 (2.59 g, 7.12 mmol) in CCl₄ (100 mL). The mixture was thenmaintained at 20° C. and irradiated with a 275 W sunlamp (Sears,#34-7105) for 2 hours over a total period of 4 hours. The irradiationwas performed by irradiating in cycles, i.e., irradiating for 10minutes, followed by 10 minutes to let the lamp cool. After the reactionwas completed, CCl₄ was removed and the residue was subjected to flashsilica gel chromatography (hexane:ethyl acetate=3) to yield the product(2.96 g, y=94.0%) as a white solid: m.p. 155°-155.5° C.; ¹ H NMR(CDCl₃), δ 0.31 (s,6H), 1.04 (S9H), 2.89 (s,4H), 4.50 (s,2H), 7.45(d,J=7.9 Hz, 1H), 7.52 (s,¹ H), 7.69 (d,J=7.9 Hz,1H); ¹³ C NMR (CDCl₃) δ-4.19, 18.22, 25.66, 27.21, 29.52, 119.97, 123.26, 126.23, 131.49,135.84, 153.98, 161.35, 169.05; IR (CHCl₃) 3018, 2932, 2859, 1772, 1742,1605, 1575, 1500, 1418, 1291, 1220 cm⁻¹ ; MS (EI) m/z (rel intensity)386 (22.0), (21.5), 329 (22.8), 327 (23.3), 272 (29.0), 270 (28.8),(43.9), 191 (42.4), 73 (100); MS (FAB,NBA matrix) m/z (rel intensity)442 (M⁺ +1, 0.42), 444 (M⁺ +3, 0.33).

3-(t (Butyldimethylsiloxyl)-4-(p-nitrophenoxy-methyl) benzoylN-hydroxysuccinimide ester (Compound 6.5)

Potassium p-nitrophenolate (0.158 g, 0.89 mmol) was added to a solutionof Compound 6.4 (0.395 g, 0.89 mmol) in freshly distilled acetonitrile(10 mL). The mixture was stirred at room temperature for 1.5 hours andthen was concentrated at reduced pressure. The product was isolated fromthe crude residue by flash silica gel chromatography to give a whitesolid (0.101 g, y=34.7%): m.p 56°-57° C. ¹ H NMR (CDCl₃) δ 0.30 (s,6H),1.01 (s,9H), 2.92 (s,4H), 5.22 (s,2H), 7.01 (dd,J=1.9, 7.3 Hz;1H), 7.54(d,J=7.2 Hz, 1H), 7.56 (s,¹ H), 7.76 (d,J=8.0 Hz, 1H), 8.23 (d,J=7.2 Hz,2H); ¹³ C NMR (CDCl₃) δ -4.23, 18.16, 25.62, 25.65, 65.75, 114.68,119.85, 123.46, 125.93, 126.08, 129.02, 133.79, 141.98, 153.38, 161.43,163.23, 168.99; IR (CHCl₃) 3018, 2931, 1772, 1742, 1694, 1517, 1420,1344, 1289, 1261, 1214 cm⁻¹ ; UV (CHCl₃) λ_(max) =305.8 nm; MS (EI) m/z(rel intensity) 443 (0.28), 386 (10.26), 328 (76.6), 172 (48.2), 135(51.6), 73 (100); MS (FAB, NBA matrix) m/z (rel intensity) 501 (M⁺ +1,0.22).

N'-(3-t-[Butyldimethylsiloxyl]-4-[p-nitrophenoxy-methyl benzoyl)glycine(Compound 6.6)

Triethylamine was added to an aqueous solution (20 mL) of glycine(0.0546 g, 0.728 mmol) to pH=12. This aqueous solution was combined atroom temperature with a solution of Compound 6.5 (0.091 g, 0.182 mmol)in acetonitrile (20 mL). The mixture was stirred for 2 minutes and thenacidified to pH 2 with 6N HCl and extracted with ether. The combinedorganic phases were evaporated and the product was purified by flashsilica gel chromatography (ethyl acetate:hexane:methanol=8:8:1) to yielda non-crystalline white solid (0.049 g, Y=58.5%): ¹ H NMR (CDCl₃) δ 0.27 (s,6H), 0.97 (s,9H), 4.25 (d,J=4.8 Hz,2H), 5.17 (s,2H), 6.94-7.00(m,3H), 7.34-7.45 (m,3H), 8.20 (d,J=9.1 Hz,2H), 10.25 (b, ¹ H); ¹³ C NMR(CDCl₃) δ -4.22, 18.19, 25.64, 41.85, 65.84, 114.68, 117.77, 119.34,125.93, 129.12, 130.44, 134.67, 141.83, 153.70, 163.54, 167.44, 173.30;MS(FAB,NBA matrix) m/z (rel intensity) 461 (M⁺ +1, 9.72).

N'-(3-t-[butyldimethylsiloxy] -4-(p-nitrophenoxy) benzoyl)glycylN-hydroxysuccinimide ester (Compound 6.7)

The method described for the synthesis of Compound 6.3 was also used toconvert Compound 6.6 to Compound 6.7. N-hydroxysuccinimide (8.3 mg,0.072 mmol) was added into a DMF (2 mL) solution of 6 (20.0 mg, 0.04mmol) and EDC (11.5 mg, 0.06 mmol) at 0° C., and the mixture was stirredfor 16 hours at 4° C. This was then diluted with brine and extractedwith ether. The ether solution was concentrated and the residue waspurified by flash silica gel chromatography (hexane:ethyl acetate=3:1)to yield the product (0.021 mg, y=86.7%) as a white solid: m.p.73°-74.5° C.; ¹ H NMR (CDCl₃) δ 0.28 (s,6H), 0.98 (s,9H), 2.85 (s,4H),4.59 (d,J=5.5 Hz,2H), 5.18 (s,2H), 6.76(t,J=5.4 Hz,1H), 6.99 (d,J= 9.2Hz,2H), 7.34 (d,J=7.9 Hz,1H), 7.38 (s,¹ H), 7.45 (d,J=7.9 Hz, 1H), 8.20(d,J=9.2 Hz,2H); ⁻⁻ C NMR (CDCl₃) δ -4.15, 18.21, 25.68, 25.59, 39.54,65.89, 114,91, 117.88 119.32, 125.94, 129.14, 130.53, 134.61, 141.91,153.74, 163.57, 165.91, 166.72, 168.48; IR (CHCl₃): 3018, 2931, 2859,2360, 1822, 1790, 1744, 1671, 1608, 1593, 1575, 1517, 1497, 1472, 1415,1372, 1344, 1259, 1218, 1216, 1210 cm⁻¹ ; UV (CHCl₃), λ_(max) =304 nm;MS (EI) m/z (rel,intensity) 517 (1.77), 460 (0.25), 436 (0.81), 403(0.91), 386 (1.2), 328 (49.64), 191 (19.29), 135 (82.84), 73 (100). MS(NH₃ /DCl, obtained on ZAB 7070 HP) exact mass 575.2173326 (M+NH₄ ⁺),calc. 575.2173.

EXAMPLE 7 Synthesis and Characterization of3-t-butyldimethyl-siloxy-4-(p-nitrophenoxy)benzamide ##STR24##3-t-Butyldimethylsiloxy-4-methylbenzamide (Compound 7.1)

3-t-Butyldimethylsiloxy-4-methylbenzoyl N-hydroxysuccinimide ester(Compound 6.3 synthesized and characterized in Example 6)) (0.206 g,0.567 mmol) was dissolved in 50 mL of a one to one mixture ofacetonitrile and water. Ammonium hydroxide (29%) was added dropwiseuntil the pH=12. After the mixture was stirred for 5 minutes at roomtemperature, it was acidified with dilute HCl to pH=2. Then etherextraction and flash silica gel chromatography (ethylacetate:hexane=2:1, Rf=0.5) gave the product as a white solid (0.142 g,y=94.7%). ¹ H NMR (CDCl₃) δ 0.24 (s,6H), 1.02 (s,9H), 2.25 (s,3H),5.60(b, ¹ H), 6.05 (b, ¹ H), 7.17-7.32 (m,3H); ¹³ C NMR (CDCl₃) δ -4.20,16.91, 18.22, 25.73, 117.67, 119.57, 130.87, 132.21, 133.61, 154.10,169.56.

3-t-Butyldimethylsiloxy-4-bromomethylbenzamide (Compound 7.2)

N-Bromosuccinimide (NBS) (0.507 g, 2.85 mmol) was added to a solution of3-t-butyldimethylsiloxy-4-methyl benzamide (Compound 7.1) in 30 mL CCl₄.The mixture was irradiated with a 275 W sunlamp (Sears, #34-7105) at 60°C. Each irradiation took 10 minutes followed by 10 minutes to let thelamp cool down. It took 2 hours of irradiation over a period of 4 hoursto complete the reaction. After the solvent was removed at reducedpressure, the residue was subjected to flash silica gel chromatographyto yield the product as a white solid (0146 g, y=81.7%). ¹ H NMR (CDCl₃)δ 0.318 (s,6H), 1.05 (s,9H), 4.51 (s,2H), 6.10 (b, 2H), 7.25 (d,J=7.8Hz, 1H), 7.32 (s,¹ H), 7.39 (d,J=7.9 Hz, 1H); ⁻⁻ C NMR (CDCl₃ ) δ 4.08,18.28, 25.76, 27.87, 117.99, 118.07, 119.49, 131.23, 132.54, 134.97,168.46.

3-t-(Butyldimethylsiloxy)-4-(p-nitrophenoxy-methyl) benzamide (Compound7.3)

Potassium p-nitro-phenolate (0.252 g, 142 mmol) was added to a solutionof Compound 7.2 (0.49 g, 1.42 mmol) in freshly distilled acetonitrile(10 mL). The mixture was stirred at room temperature for 22 hours. Afterthe solvent was removed at reduced pressure, the residue was absorbed on2 silica gel, then it was subjected to flash chromatography to give theproduct as a white solid (0.282 g, y=48.4%). ¹ H NMR (CDCl₃) δ 0.291(s,6H), 0.99 (s,9H), 5.18 (s,2H), 6.10 (b,2H), 7.00 (dd,J=2.1, 7.2Hz,2H), 7.33 (d,J=7.8Hz, 1H), 7.38 (s,¹ H), 7.45 (d,J=7.8 Hz,1H), 8.20(dd,J=2.1, 7.2 Hz,2H); ¹³ C NMR (CDCl₃) δ -4.16, 18.21, 25.65, 65.86,114.68, 117.94, 119.62, 125.95, 129.03, 130.32, 134.76, 141.80, 153.64,163.56. 168.67.

3-Hydroxy-4-hydroxyethylbenzamide (Compound 7.4)

To the solution of 3-t-butyldimethylsiloxy-4-bromomethylbenzamide(Compound 7.2) (0.101 g, 0.293, mmol) in 8 mL CH₃ CN, KF•2H₂ O (0.041 g,0.44 mmol) in 4 mL H₂ O was added dropwise. The mixture was then stirredat room temperature for half hour. After the solvent was removed, theresidue was absorbed on 500 mg silica gel. The product was obtained byflash chromatography (ethyl acetate:ethyl alcohol=10:1, Rf=0.8) as awhite solid (0.0439 g, y==90.7%). ¹ H NMR (CD₃ CN) δ 4.68 (s,2H), 5.95(b, 1H), 6.69 (b, 1H), 7.23-7.26(m,3H), 7.77 (b, 1H); ¹³ C NMR (CD₃ Cl)δ 60.66, 115.13, 119.44, 128.69, 133.07, 134.94, 156.10, 203.64.

3-Hydroxy-4-(p-nitrophenoxymethyl)benzamide (Compound 7.5)

KF•2H₂ O (0.0034 g, 0.12 mmol) was added to an acetonitrile (10 mL)solution of 3-(t-butyldimethylsiloxyl)-4-(p-nitrophenoxymethyl)benzamide(Compound 7.3) (9.8 mg, 0.024 mmol), the mixture was stirred for onehour at room temperature. The acetonitrile was removed at reducedpressure. The residue which was yellow solid was partly dissolved inacetonitrile and this acetonitrile solution was subjected to a flashsilica gel chromatography (ethyl acetate:hexane=2:1, Rf=0.12) to yield awhite solid (0.6 mg, y=9.1%): ¹ H NMR (CD₃ CN) δ 5.24 (3,2H), 6.00 (b, ¹H), 6.75 (b, ¹ H), 7.14 (d,J=0.2 Hz,2H), 7.30-7.33 (m,3H), 7.44 (d,J=7.8Hz, 1H), 7.62 (b, ¹ H), 8.20 (d,J =9.2 Hz,2H).

EXAMPLE 8 Analysis of Solvolysis of a Model Compound by ¹ H NMR

The complexity of the conjugate system renders the study of in vitroalkylation processes by ¹ H NMR difficult. Therefore a model compound(Compound 7.3 of Example 7) was synthesized which is structurallysimilar to the functional moiety of the conjugate. The solvolysis of themodel compound induced by the fluoride ion proceeded in two stages asshown in Scheme 8. ##STR25##

General Procedure for the NMR study: 429 μL of the model compound,3-t-butyldimethylsiloxy-4-(p-nitrophenoxymethyl)benzamide (Compound 7.3)(4 mM in CD₃ CN), and 71 μL of 2-[N-morpholino]ethanesulfonic acid insodium deuteroxide (MES-NaOD) buffer (70 mM, pD=6.5) were mixed in a NMRtube. The ¹ H NMR spectrum was recorded. Then 10 μL of KF (200 mM in D₂O) or LICO₄ (250 mM or 5M) was added into the system. The proton NMRspectra were recorded with the increase of time at room temperature(20=0.5° C.). For the deprotection step, the percentages of the startingmaterial at different times were calculated based on the fact that thesum of the integrations of the methylene protons peaks, δ 5.21 for thestarting material and 5.16 for the deprotection product, were unchangedwhen compared to peak of MES which was used as an internal standard. Ina similar way, for the second stage, we used the proton peaks of thep-nitrophenoxy group as the key peaks. This method is more sensitive tointegration for calculating the percentage of the solvolysis product.

The first stage of solvolysis is the deprotection of the phenol hydroxygroup on the model compound (Compound 7.3) by fluoride ion to formCompound 8.1. From proton NMR, it was observed that the decrease of themethylene proton peak of the starting material and the increase of themethylene proton peak of the product are complete in about twentyminutes. Using the integration of the methylene proton peaks, thepercentage of the starting material remaining was calculated based onthe assumption that the sum of the starting material and the product wasequal to 100%.

As shown in FIG. A, this step proceeds as a first order reaction with atime constant of 0.178 min⁻¹. It was observed that 50% conversion of thestarting material (model compound) to product (Compound 8.1) took morethan 200 hours. The methylene proton peak of the final product wasobserved at δ 4.60 ppm.

In the second stage, para-nitrophenol formed as a side product, and theintegration of the peaks for this compound were more convenient tomeasure. In this case the two doublets of the para-nitrophenol at δ 6.85ppm and 8.05 ppm were used to calculate the formation of the product(Compound 8.2). FIG. B shows the formation of the product increasingwith time with first order reaction rate constant of 0.00314 hour⁻¹.

In the presence of LiClO₄ the reaction system was kept in an NMR tube at20° C. for ten days without any obvious change in the ¹ H NMR. Thisimplies that LiClO₄ cannot promote the solvolysis of the model compoundeither by deprotection of the phenolhydroxy group or by promotion of thenucleophilic substitution at the benzylic position by water directly.

¹ H NMR monitoring did not reveal any o-quinone methide intermediateformation in the fluoride-induced solvolysis. However, the o-quinonemethide intermediate may be involved in the solvolysis process if, afterthe deprotection stage, the formation of the o-quinone methide is therate-determining step. In such a case, once the highly reactiveintermediate is formed, it is captured instantly by the solvent to formthe final solvolysis product. It is assumed that this reaction is sofast that it is not observable by NMR techniques. Nevertheless, from theNMR study, it is known that the fluoride ion is the only ion capable ofdeprotecting the phenolhydroxy group and thereby promoting furthersolvolysis. LiClO₄ was not observed to promote solvolysis, but didpromote the alkylation and cross-link in the in vitro experiments.Because of the limited conditions necessary for proton NMR studies,additional experiments (Example 9) were performed using conditionscloser to those used in the initial in vitro experiments described inExamples 1-5.

EXAMPLE 9 Kinetic Analysis of the Solvolysis of the Model Compound byUV-VIS Spectroscopy

UV-VIS spectroscopy was used to investigate the reaction in the presenceof fluoride ion, non-fluoride salts, and nucleoside. The reaction wasstudied by monitoring the formation of para-nitrophenol which has anabsorption maximum at 400 nm.

General procedure for the UV-VIS spectroscopy study: The model compound,buffer solution and additives (KF, non-fluoride salts, or nucleoside dG)were combined in a 1 mL cuvette, and maintained at 20° C. The visibleabsorbance at 400 nm was recorded with time automatically.

FIG. 6 demonstrates that a large excess of potassium fluoride does notfurther promote solvolysis of the model compound, implying thatpotassium fluoride has no effect on the second stage of solvolysis.##STR26##

FIG. 7 shows that in the presence of non-fluoride salts, the formationof para-nitrophenol is so slow as to be negligible. This is consistentwith the proton NMR study results shown in Example 8.

FIG. 8 shows that the effect of a nucleoside, deoxyguanine (dG), eitherin the presence or the absence of LiClO₄, does not promote thenucleophilic substitution of the para-nitrophenol and does not help thesolvolysis.

In summary, the model compound showed solvolysis reactivity only whenthe phenolhydroxy group was deprotected by fluoride ion. Withoutdeprotection, the model compound is apparently quite stable in theenvironment of nucleophiles such as water or nucleoside. Non-fluoridesalts were not found to enable or promote any type of nucleophilicsubstitution at the benzylic position of the model compound. Combiningthese kinetic model study results with the in vitro experimentalresults, it is reasonable to propose that the alkylation of the targetDNA by the conjugate is induced by fluoride ion via o-quinone methideintermediate formation. In the presence of non-fluoride salts, however,it is the target DNA itself which promotes the alkylation byhybridization with the conjugate to form a conformationalmicroenvironment in which the proximity between the bases on target DNAand the functional group on the conjugate is increased.

EXAMPLE 10

Additional kinds of latent bifunctional DNA alkylating agents arepossible, which spontaneously decompose, alkylate, and ultimatelycross-link duplex DNA without the aid of extracellular or intracellularactivation. In this example, a series of compounds is illustrated whichhave the inducible activity of forming two reactive electophilic centersas quinone methide intermediates and capable of cross-linking DNA asshown in Scheme 10. The positions of the two methylene groups on thearomatic ring systems may be adjusted to correspondingly adjust thedistance between the latent electrophile centers. This adjustmentenables the achievement of site specific cross-linking of duplex DNA.##STR27## The synthesis of the bifunctional compounds is illustrated inScheme 10. In this scheme, the Friedel-Craft reaction wasstraightforward and the yield was high. Proton NMR of the productmixture showed that 2,7-dimethyl-α-tetralone was the major product.Aromatization of the dimethyl tetralones by bromination, followed withrefluxing in base gives dimethyl-α-naphthols as a white solid. The majorproduct, 2,7-dimethyl-α-naphthol, was obtained as a pure crystallinesolid via recrystallization from hexane. The protection of the hydroxygroup by t-butyl dimethyl silyl chloride was straightforward withquantitative yield. Bromination of the protected 2,7-dimethyl-α-naphtholwith N-bromosuccinimide was successful, although the reaction andpurification conditions may be further optimized. ##STR28##

2,7-Dimethyl-α-tetralone (Compound 10.1a) and 2,5-dimethyl-α-tetralone(Compound 10.1b)

AlCl₃ (60.0 g,0.45 mol) was added to a freshly distilled dry toluene(200 mL) solution of α-methyl-λ-butyrolactone (10.48 g, 0.10 mol) over aperiod of 15 minutes. The reaction mixture was then stirred at 100° C.for 30 minutes, and next at 80° C. for 15 hours. It was then cooled toroom temperature and poured onto 500 crushed ice, drenched with 500 mLof concentrated hydrochloric acid. The lower aqueous layer was separatedand extracted with about 500 mL toluene. The brown organic, upper layerand the toluene extract were combined, washed successively with water,20% potassium hydroxide solution, and water, and distilled under reducedpressure to remove toluene and traces of water. Distillation of theresidue yielded (16.2 g, y=90%) dimethyl-α-tetralones: b.p. 130°-135°C./1 mm Hg.

Aromatization of the dimethyl-α-tetralones: A solution of bromine (42.35g, 0.26 mol) in 100 mL carbon disulfide (CS₂) was added dropwise to astirred solution of dimethyl-α-tetralones (38.43 g, 0.22 mol) in 200 mLCS₂ at 0° C. After stirring for an additional 3.5 hours at 4° C. thesolvent and the excess of bromine were removed at reduced pressure atroom temperature, leaving a thick dark brown oil. To this oil, 70 mLfreshly distilled N,N-dimethylaniline was added and the solution wasrefluxed for 3.5 hours under nitrogen. After it was cooled down, themixture was acidified with dilute sulfuric acid and extracted withether. After the ethereal solution was washed with 10% sodium acetatesolution and with water, it was extracted with 10% aqueous NaOHsolution. The alkaline solution was then acidified with dilute sulfuricacid and extracted with ether. The ethereal solution was washed withwater and dried with MgSO₄. After removing the ether at reducedpressure, the residue was subjected to silica gel chromatography usingpure hexane as eluant. A mixture of 2,7-dimethyl-α-naphthol (Compound10.2a) as major product and 2,5-dimethyl-α-naphthol (Compound 10.2b) asminor product was obtained as a white solid (24.58 g, y=64.7%). Pure2,7-dimethyl-α-naphthol was obtained as a crystalline solid byrecrystallization in hexane: m.p. 124°-125° C.; ¹ H NMR (CDCl₃) δ 2.40(s,3H), 2.50 (s,3H), 5.08 (b,¹ H), 7.21 (d,J=8.4 Hz, 1H), 7.29-7.33(m,2H), 7.56 (s,¹ H), 8.02 (d,J=8.55 Hz, 1H); ¹³ NMR (CDCl₃) δ 15.44,21.57, 115.48, 119.60, 120.68, 122.63, 126.70, 127.53, 129.02, 133.81,134.90, 148.58; IR(CHCl₃) 3605, 3055, 2922, 1575, 1507, 1379, 1268,1248, 1218, 1211 cm⁻¹ ; MS(EI) m/z (rel intensity) 172 (M⁺, 100) 157(25.2), 129 (38.9), 128 (42.9).

t-Butyldimethylsilyl-2,7-dimethyl-α-naphthol ether (Compound 10.3a)

2,7-dimethyl-a-naphthol (Compound 10.2a)(2.02 g, 11.8 mmol) wasdissolved in 20 mL DMF, imidazole (4.01 g, 58.9 mmol) andt-butyldimethyl chloride (4.44 g, 29.4 mmol) were then addedsuccessively. The reaction mixture was stirred at room temperature (20°C.) for 32 hours. Brine (50 mL) was added into the reaction mixture andit was extracted by ether. The ethereal solution was washed severaltimes with brine and dried with MgSO4. After removing the ether, theresidue was subjected to silica gel chromatography (hexane:ethylacetate=120:1) to yield a colorless oil as product: ¹ H NMR (CDCl₃) δ0.205 (s,6H), 1.16 (s,9H), 2.39 (s,3H), 2.51 (s,3H), 7.24-7.29(m,2H),7.36 (d,J=8.3Hz, 1H), 7.55 (s,¹ H), 7.97 (d,J=8.6 Hz,1H); ¹³ C NMR(CDCl₃) δ -3.10, 17.41, 18.79, 21.40, 26.20, 120.68, 122.00, 122.73,126.50, 126.58, 126.96, 129.61, 133.97, 134.41, 148.60; IR (CHCl₃) 3010,2954, 2930, 2860, 1592, 1565, 1498, 1473, 1410, 1355, 1260; MS(E1) m/z(rel intensity) 28 6 (M⁺, 20.7), 229 (100), 214 (36.1), 199 (31.8).

t-Butyldimethylsilyl-2,7-dibromomethylene-α-naphthol ether (Compound10.4a)

N-Bromosuccinimide (0.874 g, 4.91 mmol) was added to a solution oft-butyldimethyl-silyl-2,7-dimethyl-α-naphthol (Compound 10.3a) (0.638 g,2.33 mmol) in 25 mL CCl₄. The mixture was irradiated with a 275 Wsunlamp (Sears, #34,7105). Each irradiation took 10 minutes, followed by10 minutes to let the lamp cool down. It took 4 hours over a totalperiod of 8 hours to complete the reaction. After the by-productsuccinimide was filtered out and the solvent was removed at reducedpressure, the residue was subjected to a silica gel column which wasprerunned with drops of triethylamine and 250 mL hexane. The productobtained was a yellowish oil which contained small quantities ofover-brominated products (0.645 g, Y=65%). A small amount of pure samplewas obtained by doing silica gel chromatography again. ¹ H NMR (CDCl₃) δ0.22 (s,6H), 1.14 (s,9H), 4.63 (s,2H), 4.67 (s,2H), 7.54 (d,J=8.7Hz,1H),7.79 (s,1H), 8.08 (d,J=8.7 Hz,1H) 8.12 (m,2H), ¹³ C NMR (CDCl₃) δ -3.28,18.78, 26.02, 28.40, 33.14, 115.09, 124.58, 124.94, 127.02, 127.20,128.87, 132.53, 132.86, 137.48, 148.86,; IR (CHCl₃) 2931, 1558, 1540,1506, 1472, 1410, 1355, 1259, 1218, 1213 cm⁻¹ ; MS (EI) m/z (relintensity) 441 (M⁺, 2.1), 443 (M⁺ +2,4.0), 445 (M⁺ +4,1.2), 73 (100).

EXAMPLE 11 Experimental Procedure ##STR29##t-Butyldimethylsilyl-2,6-dimethylphenol ether (Compound 11.1)

2,6-Dimethylphenol (0.452 g, 3.7 mmol) was dissolved in 10 ml DMF, andto this imidazole (1.00 g, 14,9 mmol) and t-butyldimethyl chloride (1.12g, 7.4 mmol) were then added successively. The reaction mixture wasstirred at room temperature (20° C.) for 12 hours. Brine (30 ml) wasadded and the mixture was extracted by ether (4×30 ml). The etherealsolution was washed several times with brine and dried over MgSO₄. Afterremoving the ether, the residue was subjected to flash silica gelchromatography (hexane:ethyl acetate=120:1) to yield a colorless oil asproduct (0.873 g, y=100%): ¹ H NMR (CDCl₃) δ 0.20 (s, 6H), 1.50 (s, 9H)2.22 (s, 6H), 6.82 (t, 1H), 7.05 (d,2H).

t-Butyldimethylsilyl-2,6-di(bromoethyl)phenol ether (Compound 11.2)

N-Bromosuccinimide (0.337 g, 1.89 mmol) was added to a solution oft-butyldimethylsilyl-2,6-dimethyl phenol ether (0.224 g, 0.95 mmol) in20 ml CCl₄. The mixture was irradiated with a 275 W sunlamp (Sears,#34-7105) for a total of 160 min. The lamp was alternatively switched onand off every 10 min. Afterward, the by-product succinimide was removedby filtration and the solvent was removed at reduced pressure. Theremaining residue was subjected to silica gel chromatography(hexane:ethyl acetate=3:2) to yield a colorless oil (0.150 g, y=40%): ¹H NMR (CDCl₃) δ 0.30 (s, 6H), 1.10 (s,9H), 4.50 (s, 4H), 6.98 (t, 1H),7.38 (d, 1H).

Radiolabeling of DNA: The deoxyoligonucleotide d(AGTGCCACCTGAGGT) (0.05O.D., ca. 0.3 nmol of bases) was treated with γ-⁼ P-ATP (5.0 μL, 50 μCi,specific activity of 3000 Ci/mmol), T₄ kinase buffer (1.5 μL of 0.05MTrisHCl, pH=7.6, 0.01M MgCl₂, 5 mM DTT, 0.1 mM EDTA and 0.1 mMspermidine) and T₄ polynucleotide kinase (1 μL, 10 units) for 30 min. at37° C. The reaction mixture was diluted to 2 ml with deionized water andcentrifuged in a concentrator (Amicon, 10,000 MW cutoff) for 40 min. at4° C. (8000 rpm) to remove the excess salt and unincorporated γ-³²P-ATP. Subsequently an additional 1.5 ml water was added to the tube andthe last step was repeated. The process provided about 8.8 μCi ofphosphorylated 5'-radiolabeled oligonucleotide.

DNA cross-linking: The double helix DNA solution was prepared prior tothe reaction, 5'-⁼ P-Radiolabeled d(AGTGCCACCTGAGGT) (40 μL, 0.001 O.D.,ca. 6 pmol, 176 nCi), unlabeled d(AGTGCCACCTGACGT) and its fullycomplementary strand d(ACGTCAGGTGGCACT) (5 μL, 0.086 O.D.), 20 μLMES-NaOH buffer (10 mM, pH=7.0) and 40 μL H₂ O were placed in amicrofuge tube. The tube was heated in water bath to 85° C. and the bathand tube were allowed to cool down to room temperature. Typically, 5 μLof the above solution was mixed with 3 μL of an ethanol solution oft-butyldimethylsilyl-2,6-di(bromomethyl) phenol ether (5 mM) in amicrofuge tube. After the mixing and preincubation (various times asshown in FIG. 9), 2 μL of KF (50 mM) in water was added and it wasincubated at 37° C. for 12 hours. The reaction mixture was then dried byspeed-vac and analyzed by denaturing PAGE (20%, 7M urea) andautoradiography.

FIG. 9 shows an autoradiogram of denaturing polyacrylamide gel (20%)used to identify the cross-link product of duplex DNA byt-butyldimethyl-2,5-dibromomethylenephenol ether (Compound 11.2). Lane1: duplex DNA(3 μM)+KF(10 mM): Lane 2: duplex DNA(3 μM) alone: Lane 3:duplex DNA(3 μM)+Compound 11.2 (1.5 mM); Lane 4: duplex DNA(3μM)+Compound 11.2 (1.5 mM), 12 hours pre-incubation at 25° C., additionof KF (10 mM), 30 min. incubation at 37° C.; Lane 5: duplex DNA(3μM)+Compound 11.2 (1.5 mM), no pre-incubation, addition of KF (10 mM),12 hours incubation at 37° C.; Lane 6: duplex DNA(3 μM)+Compound 11.2(1.5 mM), 12 hours pre-incubation at 25° C., addition of KF (10 mM), 30min. incubation at 25° C.; Lane 7: duplex DNA(3 μM)+Compound 11.2 (1.5mM), no pre-incubation, addition of KF (10 mM), 12 hours incubation at25° C.

Thus, while we have described what are presently the preferredembodiments of the present invention, other and further changes andmodifications could be made thereto without departing from the scope ofthe invention, and it is intended by the inventor herein to claim allsuch changes and modifications.

We claim:
 1. A silyloxy aromatic probe composition having the formula:##STR30## wherein one of R₁, R₂, R₃, R₄ and R₅ is --OSiR₆ R₇ R₈ ;whereinwhen R₁ =--OSiR₆ R₇ R₈, then R₂ and/or R₄ is =--CR₉ R₁₀ X; when R₂=--OSiR₆ R₇ R₈, then R₁, R₃ and/or R₅ is =--CR₉ R₁₀ X; when R₃ =--OSiR₆R₇ R₈, then R₂ and/or R₄ is =--CR₉ R₁₀ X; when R₄ =--OSiR₆ R₇ R₈, thenR₁, R₃ and/or R₅ is =--CR₉ R₁₀ X; when R₅ =--OSiR₆ R₇ R₈, then R₂ and/orR₄ is =--CR₉ R₁₀ X; any remaining R₁ -R₅ substituents that are not--OSiR₆ R₇ R₈ or --CR₉ R₁₀ X are hydrogen atoms; wherein said R₆, R₇, R₈=alkyl or aromatic groups; wherein R₉ and R₁₀ is an aliphatic or alkylgroup; X=leaving group; L is a linking group for attachment to a probewhich may be positioned at any carbon atom of the ring; and Pr is aprobe for binding to DNA or RNA.
 2. A silyloxy aromatic probe accordingto claim 1,wherein said silyloxy aromatic probe alkylates DNA or RNA inresponse to ionic activation; and wherein X is selected from the groupconsisting of Cl, F, I, --OCOR, --OH, --OSO₂ CH₃, --OSO₂ C₆ H₄ CH₃ --p,--OR, --OCONHR, --OCONHCH₂ CH₂ R, Br, --OC₆ H₅, --OC₆ H₄ NO₂, and --SC₆H₅.
 3. A silyloxy aromatic probe according to claim 2, wherein Lcomprises a chain --R₁₁ --R₁₂ --R₁₃, in which R₁₁ is selected from thegroup consisting of --NH----S--, --O--, and --CH₂ --, in which R₁₂comprises a stable spacer group between R₁₁ and R₁₃, and in which R₁₃ isselected from the group consisting of --NH₂ --, --SH, --OH and --COOH;andwherein Pr is a probe that includes a localizing moiety selected fromthe group consisting of an oligonucleotide, protein, intercalatingmoiety, or other molecule that localizes to DNA or RNA.
 4. A silyloxyaromatic probe according to claim 3, wherein R₆, R₇ and R₈ comprises at-butyl moiety.
 5. A silyloxy aromatic probe according to claim 3,wherein Pr is an oligonucleotide.
 6. A silyloxy aromatic probe accordingto claim 3, wherein Pr is a protein.
 7. A silyloxy aromatic probeaccording to claim 5, wherein Pr is a DNA strand.
 8. A silyloxy aromaticprobe according to claim 3, wherein Pr is an intercalating moiety forintercalating into DNA or RNA.
 9. A silyloxy aromatic probe according toclaim 5, wherein said oligonucleotide is linked to L by its 5' terminus.10. A silyloxy aromatic probe according to claim 5, wherein theoligonucleotide is linked to L by its 3' terminus.
 11. A silyloxyaromatic probe according to claim 5, further comprising a modified baseon said oligonucleotide suitable for linking to R₁₂ at said modifiedbase.
 12. A silyloxy aromatic probe according to claim 5, furthercomprising a modified phosphoribose backbone on said oligonucleotidesuitable for linking to R₁₂ at said modified phosphodeoxyribosebackbone.
 13. A silyloxy aromatic probe having the formula: ##STR31##wherein R₂ =--OSiR₆ R₇ R₈, R₃ =--CR₉ R₁₀ X R₁,R₄ and R₅ =hydrogen;saidR₆, R₇, R₈ =alkyl or aromatic groups; said R₉ and R₁₀ is an aliphatic oralkyl group, X =leaving group; L is a linking group for attachment to aprobe which may be positioned at any carbon atom of the ring; and Pr isa probe for binding to DNA or RNA.
 14. A silyloxy aromatic probeaccording to claim 13, wherein L comprises a chain --R₁₁ --R₁₂ --R₁₃ --,in which R₁₁ is selected from the group consisting of --NH--, --S--,--O-- and --CH₂ --, in which R₁₂ comprises a stable spacer group betweenR₁₁ and R₁₃, and in which R₁₃ is selected from the group consisting of--NH₂ --, --SH, --OH and --COOH; and p1 wherein Pr is a probe thatincludes a localizing moiety, selected from the group consisting of anoligonucleotide, protein, intercalating moiety, or other molecule thatlocalizes to DNA or RNA.
 15. A silyloxy aromatic probe according toclaim 14, wherein R₆ and R₇ comprise methyl groups and R₈ comprises at-butyl moiety.
 16. A silyloxy aromatic probe according to claim 14,wherein Pr is an oligonucleotide.
 17. A silyloxy aromatic probeaccording to claim 14, wherein Pr is a protein.
 18. A silyloxy aromaticprobe according to claim 14, wherein Pr is a DNA strand.
 19. A silyloxyaromatic probe according to claim 14, wherein Pr is an intercalatingmoiety for intercalating into DNA or RNA.
 20. A silyloxy aromatic probeaccording to claim 16, wherein said oligonucleotide is linked to L byits 5' terminus.
 21. A silyloxy aromatic probe according to claim 16,wherein said oligonucleotide is linked to L by its 3' terminus.
 22. Asilyloxy aromatic probe according to claim 16, further comprising amodified base on said oligonucleotide suitable for linking to R₁₂ atsaid modified base.
 23. A silyloxy aromatic probe according to claim 16,further comprising a modified phosphoribose backbone on saidoligonucleotide suitable for linking to R₁₂ at said modifiedphosphoribose backbone.
 24. A silyloxy aromatic probe recited in claim15, wherein X comprises --OC₆ H₄ NO₄.
 25. A process for alkylating DNAor RNA, comprising steps of:a) providing a probe for binding to DNA orRNA; b) providing a silyloxy benzene derivative for linking to theprobe; c) linking the probe to the silyloxy benzene derivative to form atargeted alkylating agent; d) introducing the targeted alkylating agentto an in vitro system containing DNA or RNA, whereby the probeassociates with the DNA or RNA, localizing the linked silyloxy aromaticderivative near the DNA or RNA; and e) activating the targetedalkylating agent, thereby causing covalent binding between the linkedaromatic derivative and the DNA or RNA proximal to the association siteof the probe with the DNA or RNA.
 26. A process as recited in claimwherein the targeted alkylating agent has the molecular formula:##STR32## wherein one of R₁, R₂, R₃, R₄ and R₅ is --OSiR₆ R₇ R₈ ;whereinwhen R₁ =--OSiR₆ R₇ R₈, then R₂ and/or R₄ is =--CR₉ R₁₀ X; when R₂=--OSiR₆ R₇ R₈, then R₁, R₃ and/or R₅ is =--CR₉ R₁₀ X; when R₃ =--OSiR₆R₇ R₈, then R₂ and/or R₄ is =--CR₉ R₁₀ X; when R₄ =--OSiR₆ R₇ R₈, thenR₁, R₃ and/or R₅ is =--CR₉ R₁₀ X; when R₅ =--OSiR₆ R₇ R₈, then R₂ and/orR₄ is =--CR₉ R₁₀ X; wherein any remaining R₁ -R₅ substituents that arenot --OSiR₆ R₇ R₈ or --CR₉ R₁₀ X are hydrogen atoms; wherein said R₆,R₇, R₈ =alkyl or aromatic groups; said R₉ and R₁₀ is an aliphatic oralkyl group; X=leaving group; L is a linking group for attachment to aprobe which may be positioned at any carbon atom of the ring; and Pr isa probe for binding to DNA or RNA.
 27. A process as recited in claim 26,wherein said linking step (c) further comprises adapting the silyloxyaromatic derivative by the addition of an acidic linking group suitablymodified for linking the silyloxy aromatic derivative to the probemolecule.
 28. A process as recited in claim 27, wherein said probe is anoligonucleotide, and further comprising the step of:suitably modifying abase of said oligonucleotide probe for linking to L at the modifiedbase.
 29. A process as recited in claim 27, wherein said probe is anoligonucleotide, and further comprising the step of:suitably modifying aphosphoribose backbone of said oligonucleotide probe for linking to L atthe modified phosphoribose backbone.
 30. A process as recited in claim27, wherein said activating step comprises introduction of an ionicactivating signal.
 31. A process as recited in claim 27, wherein saidadapting step further includes brominating the X group, and saidactivating step comprises activation with an ionic signal.
 32. A processrecited in claim 31, wherein said brominating step is followed bysubstituting the bromine by molecules selected from the group consistingof Cl, F, I, --OCOR, OH, --OSO₂ CH₃, --OSO₂ C₆ H₄ CH₃ --p, --OR,--OCONHR, --OCONHCH₂ CH₂ R, Br, --OC₆ H₅, --OC₆ H₄ NO₂, and --SC₆ H₅.33. A process as recited in claim 32, wherein L comprises a chain --R₁₁R₁₂ --R₁₃ --, in which R₁₁ is selected from the group consisting of--NH--, --S--, --O--, and --CH₂ --, in which R₁₂ comprises a stablespacer group between R₁₁ and R₁₃, and in which R₁₃ is selected from thegroup consisting of --NH₂, --SH, --OH and --COOH; andwherein Pr is aprobe that includes a localizing moiety selected from the groupconsisting of an oligonucleotide, protein, intercalating moiety, orother molecule that localizes to DNA or RNA.
 34. A silyloxy aromaticcompound, comprising a substituted benzene ring, wherein a position onthe benzene ring is occupied by an --OSiR₆ R₇ R₈ group and at least oneposition on the benzene ring in conjugation with the --OSiR₆ R₇ R₈ groupis occupied by a --CR₉ R₁₀ X group, in whichR₆, R₇, and R₈ are selectedfrom the group consisting of alkyl and aromatic groups; R₉ and R₁₀ areselected from the group consisting of alkyl and aromatic groups; and Xis a leaving group.
 35. The silyloxy aromatic compound of claim 34,wherein two positions on the benzene ring in conjugation with the--OSiR₆ R7R₈ are occupied by --CR₉ R₁₀ X group.
 36. The silyloxyaromatic compound of claim 34, wherein a heteroatom is present withinthe benzene ring without destroying the conjugation between the --OSiR₆R₇ R₈ and --CR₉ R₁₀ X groups.
 37. The siloxy aromatic compound of claim36, wherein said heteroatom is selected from the group consisting of Oand N.
 38. The silyloxy aromatic compound according to claim 34, whereinsaid R₆ and R₇ comprise methyl groups and R₈ comprises a t-butyl moiety.39. The silyloxy aromatic compound according to claim 34, wherein --X isselected from the group consisting of F, Cl, Br, I, --OCOR, OH, --OSO₂CH₃, --OSO₂ C₆ H₄ CH₃ --p, --OR, --OCONHR, --OCONHCH₂ CH₂ R, --OC₆ H₅,--OC₆ H₄ NO₂, and --SC₆ H₅.
 40. The silyloxy aromatic compound accordingto claim 34, wherein X is selected from the group consisting of OC₆ H₄NO₂ and Br.
 41. The silyloxy aromatic compound according to claim 34,wherein the silyloxy aromatic compound alkylates DNA or RNA in responseto ionic activation.
 42. The silyloxy aromatic compound according toclaim 34, wherein a position on the benzene ring is occupied by a--L--Pr group, in which L is a linking group for attachment of thearomatic ring system to a probe; and Pr is a probe for binding to DNA orRNA.
 43. The silyloxy aromatic compound according to claim 42, wherein Lcomprises a chain --R₁₁ --R₁₂ --R₁₃ --, in whichR₁₁ is selected from thegroup consisting of --NH--, --S--, --O--, and --CH₂ --; R₁₂ comprises astable spacer group between R₁₁ and R₁₃ ; R₁₃ is selected from the groupconsisting of --NH₂, --SH--, --OH, and --COOH.
 44. The silyloxy aromaticcompound according to claim 42, wherein Pr is a probe that includes alocalizing moiety selected from the group consisting of anoligonucleotide, protein, intercalating moiety, or other molecule thatlocalizes to DNA or RNA.
 45. The silyloxy aromatic compound according toclaim 44, wherein the localizing moiety non-specifically localizes toDNA or RNA.
 46. The silyloxy aromatic compound according to claim 44,wherein the localizing moiety specifically localizes to DNA or RNA. 47.The silyloxy aromatic compound according to claim 44, wherein thelocalizing moiety is an oligonucleotide.
 48. The silyloxy aromaticcompound according to claim 47, wherein the localizing moiety is aprotein.
 49. The silyloxy aromatic compound according to claim 44,wherein the localizing moiety is a DNA strand.
 50. The silyloxy aromaticcompound according to claim 44, wherein the localizing moiety is anintercalating moiety for intercalating into DNA or RNA.